Compositions and Methods to Elicit Immune Responses Against Pathogenic Organisms Using Yeast Based Vaccines

ABSTRACT

Embodiments of the present invention illustrate methods of treating and preventing infection due to a pathogen such as a fungal pathogen. In particular, the present invention relates to compositions and methods for vaccinations against or treatment for a fungal organism in a non-immunocompromised or immunocompromised subject.

RELATED APPLICATIONS

This application is a continuation of copending U.S. patent applicationSer. No. 11/610,457, filed Dec. 13, 2006, which is incorporated hereinby reference in its entirety, and which claims the benefit of priorityunder 35 U.S.C. §119(e) of U.S. provisional application No. 60/750,029,filed on Dec. 13, 2005, which is incorporated herein by reference in itsentirety, and of U.S. provisional application No. 60/817,300, filed onJun. 28, 2006, which is incorporated herein by reference in itsentirety.

FEDERALLY FUNDED RESEARCH

The studies disclosed herein were supported in part by grants 1 R43 AI052632-01A1, 1 R43 AI054020-01A1 and 1 R41 AI062482-01 from the SmallBusiness Innovation Research Program (SBIR) and STTR Program of theNational Institutes of Health. The U.S. government may have certainrights to practice the subject invention.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted electronically asa text file by EFS-Web. The text file, named “3923-28-1_ST25”, has asize in bytes of 47 KB, and was recorded on 30 Sep. 2009. Theinformation contained in the text file is incorporated herein byreference in its entirety pursuant to 37 CFR §1.52(e)(5).

FIELD

The present invention provides for methods, compositions and kits forreducing a fungal infection and/or inducing a protective or therapeuticresponse against a pathogenic or non-pathogenic organism.

BACKGROUND

Many conditions in humans and animals are caused by fungal infectionsand current therapeutics for the prevention of and treatments for theseinfections are largely ineffective. Coccidioidomycosis, also known asSan Joaquin Valley Fever, is a fungal disease caused by Coccidioidesimmitis that is endemic in portions of Southern Arizona, centralCalifornia, southern New Mexico and west Texas. At least 100,000 newcases are reported each year. The migration of not only permanentresidents, but also agricultural workers to these areas increasesexposure to C. immitis spores that lie dormant in the soil, and as thesoil is disturbed, the spores become airborne and are inhaled. Once inthe lungs, the arthroconidia transform into spherules. An acuterespiratory infection occurs between seven days to three weeks afterexposure and often resolves rapidly. However, in a significant number ofcases, chronic pulmonary conditions or dissemination to the meninges,bones, and joints can result, leading to acute, life-threateningdisease. One population, migrant laborers, is exposed to C. immitis andare a highly susceptible to get infected. A variety of approaches havebeen used to fight coccidioidomycosis, including soil treatments, butonly a vaccine can completely eliminate this “emerging disease.”

Another pathogen, Cryptococcus neoformans is an encapsulated pathogenicyeast that causes pulmonary infections and meningoencephalitis in humansand other animals. During the last 20 years, there has been a dramaticincrease in the incidence of cryptococcosis throughout the world thatmirrors the increase in not only HIV infections, but also in the growingnumber of immunocompromised patients. Loss of CD4⁺ T cells predisposespatients to progressive infection with C. neoformans—this emphasizes therole of cell-mediated immunity in host resistance.

C. neoformans is a basidiomycetous fungus that is generally isolated asa haploid yeast, although diploids have been identified in nature. Thereare two mating types that can undergo recognition and fusion, and form amycelium. A structure called a basidium is made and spores are producedfrom the surface of the basidium. Under specific conditions, haploidcells can undergo sporulation. Recently, a stable diploid was shown togrow as a yeast at 37° C. and formed hyphae and produced spores at 24°C. C. neoformans has been the subject of many studies, the var. grubiistrain H99, as the source of the candidate immunogens. Var. grubii areserotype A strains, have a worldwide distribution, and are the mostcommon variety to cause disease in the United States. Strain H99 hasseveral advantages. First, molecular genetic analysis, including genedeletion and allelic replacement experiments, are well established inH99. Second, the genome sequence of H99 has been determined. The randomshotgun and assembly phases of the genome are complete and the assembledgenome has been released. Third, a congenic mating partner for H99 hasrecently been developed. Lastly, several reproducible animal models havebeen well developed to assess virulence for this strain.

Cryptococcosis is a disease that is acquired by inhalation of theorganism from the soil or avian droppings into the lungs. Bothimmunocompetent and immunocompromised patients can be infected with theorganism. In immunocompetent patients, the disease is usually containedin a lung granuloma and induces an antibody response. In contrast,individuals whose cellular immunity has been compromised by viralinfection, suppression due to tissue transplantation, or anti-neoplasticchemotherapy are particularly susceptible to disseminated disease,followed by often-fatal meningoencephalitis. For example, an estimated7-10% of AIDS patients acquire cryptococcosis during the course of theirHIV disease. In compromised hosts, patients with cryptococcosisgenerally present with symptoms of meningitis such as fever, headache,and malaise. Cryptococcal pneumonia is the next most frequentmanifestation of cryptococcosis in immunocompromised patients. It occursas a primary infection in approximately 4% of cases and is associatedwith general symptoms of pneumonia such as fever, cough, pleuritic chestpain, and/or dyspnea.

Aspergillus fumigatus is a ubiquitous spore-bearing fungus that causesmultiple diseases in humans. These include allergic asthma,aspergillomas, and invasive pulmonary disease of hosts with predisposingunderlying conditions. In the United States in 1996, there were anestimated 10,190 aspergillosis-related hospitalizations; these resultedin 1,970 deaths, 176,300 hospital days, and $633 million in costs. Theaverage hospitalization lasted 17.3 days and cost ˜$62,000. Althoughaspergillosis-related hospitalizations account for a small percentage ofhospitalizations in the United States, patients hospitalized with thecondition have lengthy hospital stays and high mortality rates. The highmortality rates (in some instances over 90%) are due, in part, to thelack of rapid and sensitive diagnostic tests (all too often thediagnosis is made post mortem), as well as the lack of effectiveanti-fungal therapeutics.

The most common species of Aspergillus causing invasive disease includeA. fumigatus, A. flavus, A. niger, A. terreus and A. nidulans. A.fumigatus is the most frequently found fungus in airborne spore surveys.The organism grows in a variety of environments including air ducts,houseplant soil, compost piles, and at a wide range of temperatures,from ±12° C. to 55° C. Of the genus Aspergillus, A. fumigatus is themost common pathogen of man.

Currently, there are a number of anti-fungal vaccine and anti-fungaltreatment effort that use a variety of approaches including selectedrecombinantly-expressed antigens. Safe and effective vaccines againstfungal organisms as well as against other similar pathogens are needed.

SUMMARY

Embodiments of the present invention concern methods and compositionsfor reducing the onset of, preventing or as treatment for a fungalinfection. In accordance with these embodiments, a composition derivedat least in part from non-viable fungal cells can be used to vaccinate asubject against or treat for a fungal infection. In certain embodiments,fungal cells were heated at a temperature of about 56° C. for about onehour. In other embodiments, fungal cells were heated at temperaturesranging from about 50° C. to about 70° C. for from about 30 minutes toabout 2 hours. Time and temperature may vary depending on the fungalorganism, as well as, other factors such as the type of vaccine ortherapeutic desired.

Certain embodiments of the present invention concern non-viable fungalcell compositions including fungal cells that were heated at atemperature of about 56° C. for about one hour, or a derivative thereof.Other particular embodiments concern vaccines, wherein the vaccinesinclude a composition derived from non-viable fungal cells and whereinfungal cells were heated at a temperature of about 56° C. for about onehour. In accordance with these embodiments, the fungal cells can betransgenic, non-transgenic or a combination thereof. The vaccinecompositions contemplated herein are capable of inducing an immuneresponse against viable and non-viable fungal cells when administered toa subject. Fungal cells can include, but are not limited to,Saccharomyces spp., Aspergillus spp. Cryptococcus spp., Coccidioidesspp., Neurospora spp., Histoplasma spp., Blastomyces spp., and acombination thereof. In other embodiments, the fungal cells may furthercomprise a tag. In certain exemplary transgenic vaccines, compositionscan include fungal cells transformed with an oligonucleotide, a peptide,a protein, a chimeric molecule or combination thereof. In addition,transgenic fungal cells can contain one or more virulence factor.

In some embodiments, a derivative of a non-viable fungal composition canbe an active portion of non-viable fungal cells or a fraction thereofthat is capable of inducing an immune response when introduced to asubject. In accordance with these embodiments, the derivative can beused a prophylactic against, prevention of and/or treatment for a fungalinfection.

In certain embodiments of the present invention, the fungal cellscomprise transgenic, non-transgenic or a combination thereof. In otherembodiments, the fungal cells can include, but are not limited to,Saccharomyces spp., Aspergillus spp., Cryptococcus spp., Coccidioidesspp., Neurospora spp., Histoplasma spp., Blastomyces spp., and acombination thereof.

In certain particular embodiments, Saccharomyces spp. cells can bespores, vegetative cells, germlings, or a combination thereof. In otherembodiments, the Neurospora spp. cells can be vegetative hyphae, aerialhyphae, macroconidia, germinating macroconidia, microconidia,germinating microconidia, ascospores, germinating ascospores, or acombination thereof. In other embodiments, the Aspergllus spp. cells canbe vegetative hyphae, conidia, or germinating conidia, or combinationsthereof.

In certain embodiments, Coccidioides spp. cells can be arthoconidia,germinating arthroconidia, hyphae, spherules, germinating spherules,endospores, germinating endospores, or a combination thereof. In otherparticular embodiments, Cryptococcus spp. cells can be yeast cells.

In other embodiments, Histoplasma spp. cells can be yeast cells,vegetative hyphae, microcondia, germinating microconidia, macroconidia,germinating macroconidia, or combinations thereof.

In other embodiments, Blastomyces spp. cells can be yeasts, hyphae,blastoconidia, germinating blastoconidia or combinations thereof. Insome embodiments of the present invention, the fungal cells may furtherinclude a tag. In other particular embodiments, transgenic fungal cellscan contain at least one oligonucleotide. In certain examples, theoligonucleotide can encode a peptide or a protein. Alternatively, thetransgenic fungal cells contain at least one virulence factor.

Embodiments of the present invention concern administering to a subjecta vaccine that includes, at least in part, non-viable fungal cells. Incertain embodiments, vaccines of the present invention induce an immuneresponse in the subject directed against one or more fungal organisms.In accordance with these embodiments, the subject is a mammal, bird orreptile. In one particular embodiment, the subject is a human. Incertain examples, the vaccine can be administered in a single dose, afew doses or multiple doses to reduce the onset of a fungal infection,preventing and/or treating a fungal infection. In other embodiments, thevaccination can occur yearly, monthly, or weekly or even more frequentdepending on need.

In other embodiments, a subject having a fungal infection can be treatedprophylactically to reduce progression of the fungal infection and oreliminate the infection.

In certain embodiments of the present invention, the subject is a humanpredisposed to a fungal infection or a fungal organism (e.g.,predisposed can mean at risk of exposure or having been exposed to afungal infection or fungal organism). In other embodiments, the subjectis an immunocompromised subject. In accordance with this embodiment, theimmunocompromised subject is a subject having a viral infection, tissuetransplantation, anti-neoplastic chemotherapy or combination thereof.

Other embodiments of the present invention concern kits for treatingand/or preventing a fungal infection. In accordance with theseembodiments, the kits can include non-viable fungal cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the presentinvention. The embodiments may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1 represents exemplary growth curves of yeast containing a geneencoding an antigen grown in the presence of various concentrations ofCuSO₄.

FIG. 2 illustrates an exemplary growth curve of yeast cells containingand expressing an antigen protein.

FIG. 3 illustrates an exemplary schematic of a method to test a vaccinedisclosed herein.

FIG. 4 illustrates exemplary survival curves for mice infected in vitrowith A. fumigatus.

FIG. 5. represents an exemplary electrophoresis gel of separated nucleicacid sequences from PCR reactions.

FIG. 6. illustrates an exemplary schematic of a method to test a vaccinedisclosed herein.

FIG. 7 illustrates exemplary survival curves for mice infected in vitrowith C immitis.

FIG. 8 represents an exemplary Western blot of separated lysed yeastcells containing and expressing genes for hemolysin, aspf2 orantigen-2/PRA.

FIG. 9 illustrates an exemplary schematic of a method to test a vaccinedisclosed herein.

FIG. 10. represents an exemplary electrophoresis gel of separatedmolecules of yeast lysates containing and expressing the gene encodingChitin deacetylase and Laccase.

FIG. 11 illustrates an exemplary survival curves for mice infected invitro. with Cryptococcosis neoformans

FIG. 12 illustrates an exemplary scattergram of protective efficacy ofAspergillus hemolysin antigen expressed in Saccharomyces spp. againstsystemic aspergillosis in mice.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, “about” can mean plus or minus ten percent.

DETAILED DESCRIPTION

In the following sections, various exemplary compositions and methodsare described in order to detail various embodiments of the invention.It will be obvious to one skilled in the art that practicing the variousembodiments does not require the employment of all or even some of thespecific details outlined herein, but rather that sequences chosen,proteins selected, samples, concentrations, times and other specificdetails may be modified through routine experimentation. In some cases,well-known methods or components have not been included in thedescription.

Embodiments of the present invention provide for compositions andmethods for reducing the onset of, preventing and/or treating a fungalinfection. In particular embodiments, methods and compositions whenadministered to a subject are capable of inducing a protective immuneresponse against a target. For example, these embodiments include afungal pathogen. In accordance with these embodiments a fungalpathogenic organism includes, but is not limited to, Aspergillus spp.,Cryptococcus spp., and Coccidioides spp. In exemplary embodiments, suchpathogenic fungus may include, but are not limited to, Aspergillusfumigatus, A. flavus, A. niger, A. terreus, A. nidulans, Coccidioidesimmitis, Coccidioides posadasii and Cryptococcus neoformans. The immuneresponse may be induced by injection of a fungal-based vaccine. Suchvaccines may comprise, but are not limited to, suspensions, solutions,extracts, homogenates, precipitates and/or other processed orunprocessed forms of Saccharomyces cerevisiae. Although in exemplaryembodiments the vaccines may comprise yeast transformed with geneticmarkers, such as a c-myc tag sequence. In one embodiment, the vaccinesmay include a non-transgenic fungus or fungi. In one particularembodiment, the vaccines may include non-transgenic or transgenic yeast.

Embodiments of the present invention provide for combination treatmentsof a subject in need of a vaccine against an organism such as apathogenic fungal organism. In accordance with these embodiments, anyvaccine treatment detailed herein may be combined with, but is notlimited to, treatments for cryptococcosis, aspergillosis, orcoccidoidomycosis such as amphotericin B or the azoles, fluconazole oritraconazole, 5-flucytosine, voriconazole, and fluconazole. In anotherembodiment, other treatments may include other antifungal treatments,such as any of the echinocandin class antifungals or any otherantifungal agent.

It is contemplated herein that any of the vaccines disclosed may beadministered to a subject having or suspected of developing a conditiondue to exposure or potential exposure to a fungal pathogen. In oneembodiment, a subject may include a human. In other embodiments asubject may include a human, other mammals, birds and reptiles. In yetanother embodiment, the subject can include a domesticated animal. Anyof the methods disclosed herein may be used in combination with otherantifungal agents or therapies to treat a subject in order to achievethe desired results.

In another embodiment, it is contemplated that a subject can bevaccinated and/or treated using a composition against a fungal pathogendisclosed herein before infection, during infection, after infection orcombination thereof. In one particular embodiment, a compositiondisclosed herein may be used to vaccinate a subject against a particularfungal organism to reduce the risk of a fungal infection in the subject.In another embodiment, a composition disclosed herein may be used tovaccinate a subject against one or more fungal organisms to reduce therisk of a fungal infection in the subject. In certain embodiments, avaccine containing non-viable fungal cells of one fungal type whenadministered to a subject may reduce the onset or progression of fungalinfection of one or more additional fungal types in the subject.

In certain embodiments of the present invention, a vaccine or treatmentof use in a subject can contain non-viable fungal cells where the fungalcells are transgenic, non-transgenic or a combination thereof.Transgenic cells of the present invention may include anoligonucleotide. In certain examples, the oligonucleotide can encode oneor more peptides or proteins.

Advantages

Approaches disclosed herein have several advantages over current fungalvaccines. First, the fungal cells expressing each fungal protein may beengineered so that it is not secreted but rather is retainedintracellularly. This likely obviates the need for large amounts ofrecombinant fungal antigen protein to be produced and purified,ultimately under cGMP conditions. Second, fungal cells are easilytransformed and manipulated. Third, putative antigens can be testedrather inexpensively and ineffective antigens can be eliminated quickly.Fourth, whole, heat-killed yeast as a vaccine delivery vehicle is noveland presents antigens directly to dendritic cells to induce the immunesystem. Fifth, several individual antigen-expressing yeast strains canbe mixed to form a vaccine, a possibility not readily available tocurrent vaccine projects. Sixth, the yeast-based vaccine technology haseach of the characteristics of a successful vaccine: generates aproductive immune response; not neutralized; capable of generating aresponse to multiple epitopes; and stable and easy to manufacture.

One example of a beneficial therapeutic or a vaccine might consist of avector with no pathogenic potential that can deliver several antigens toantigen-presenting cells (APCs). The use of recombinant yeast proves tobe an ideal vector for vaccine development. In one embodiment, a vaccinecontemplated herein could consist of a vector with no pathogenicpotential that can deliver several antigens to APCs. In this regard, theuse of recombinant yeast proves to be an ideal vector for vaccinedevelopment. The yeast Saccharomyces cerevisiae is avidly taken up byprofessional APCs, such as neutrophils, macrophages and dendritic cells.Multiple antigens can be engineered for expression within a single yeastformulation, and these formulations share many advantages with DNAvaccines, including ease of construction, low expense of mass productionand biological stability. Unlike DNA vaccines, yeast-based vaccineformulations do not require extensive purification to remove potentiallytoxic contaminants. Furthermore, while the FDA has not evaluated yeastas a vaccine vector, the organism S. cerevisiae is designated by the FDAas GRAS (Generally Regarded As Safe, FDA Proposed Rule 62FR18938, Apr.17, 1997). As described below, the heterologous proteins expressed inrecombinant yeast serve as antigens that elicit CD8+CTL-mediated immuneresponses in vitro and in vivo. In animal trials as a tumor vaccine, theyeast formulation was successful at protecting vaccinated animals fromtumor growth (refer to FIGS. 4, 7, 11 and 12 as examples of animal modeltesting).

In another embodiment, it is contemplated that additional immunogens forin vivo efficacy may be tested using methods disclosed herein.

In addition, it is contemplated that the immunogens may be of a singleformulation or a combination formulation. To test a formulation,immunocompetent animals or immuncompromised animals may be used in orderto assess what formulation may be used to treat a subject. The test caninclude vaccination and then a challenge of the vaccination. In anotherembodiment, the appropriate dose of any disclosed therapeutic or vaccinemay be determined by the methods disclosed herein including, but notlimited to optimum formulation, the appropriate number of treatments orvaccinations and the optimum time for treatment or vaccinations in orderto instill maximum treatment or protection against a given organism.

In certain embodiments, methods and compositions disclosed herein aredirected toward making and using an anti-fungal vaccine or anti-fungaltreatment. Fungal organisms targeted in the present invention include,but are not limited to, Saccharomyces spp., Aspergillus spp.Cryptococcus spp., Coccidioides spp., Neurospora spp., Histoplasma spp.,Blastomyces spp., and a combination thereof.

Aspergillus

In certain embodiments of the present invention, methods, compositionsand treatments for a fungal organism infection can concern treatment foror prevention of an Aspergillus associated-condition. The most commonspecies of Aspergillus causing invasive disease include A. fumigatus, A.flavus, A. niger, A. terreus and A. nidulans. A. fumigatus is the mostfrequently found fungus in airborne spore surveys. A. fumigatuspneumonia and systemic aspergillosis occur primarily in patients whohave immunosuppression or T-cell or phagocytic impairment. Theimmunodeficiency detected in these patients may be congenital, acquiredor iatrogenic. Patients with chronic granulomatous diseases, neutrophildysfunction, and with severe immunodeficiency are at risk for thedevelopment of this predominantly fatal infection. Although no importantprotective antibody response was detected, CD4+ Th1 cytokines appear tobe important in rendering protection in these patients. In oneembodiment of the present invention, a subject having or suspected ofdeveloping a disease derived from an Aspergillus species can beadministered a composition disclosed herein. In one particularembodiment, a subject having or suspected of developing a diseasederived from an Aspergillus species can be administered a compositionincluding heated, transformed or non-transformed fungi for example,heated Aspergillus fungi.

Three main types of diseases are attributed to A. fumigatus; theseinclude asthma, aspergillomas and invasive aspergillosis. The mostserious disease involves invasion of hosts with predisposing underlyingconditions. Patients undergoing organ transplants, bone marrowtransplants, leukemics, or cancer chemotherapy are particularly at riskfor invasive aspergillosis. Aspergillosis is often diagnosed when thereis an unexplained pulmonary infiltrate, a patient is unresponsive toantibacterials and/or there is a fever of unknown origin. The prognosisfor patients with invasive disease is high (mortality rates >50%) due tothe lack of a rapid diagnostic test confirming A. fumigatus infectionand the lack of safe and effective antifungal drugs.

Coccidioides

Coccidioidomycosis, also known as San Joaquin Valley Fever, is a fungaldisease caused by Coccidioides immitis that is endemic in portions ofSouthern Arizona, central California, southern New Mexico and westTexas. At least 100,000 new cases are reported each year. The migrationof not only permanent residents, but also agricultural workers to theseareas increases exposure to Coccidioides spp. spores that lie dormant inthe soil, and as the soil is disturbed, the spores become airborne andare inhaled. Once in the lungs, the arthroconidia transform intospherules. An acute respiratory infection occurs between seven days tothree weeks after exposure and often resolves rapidly. However, in asignificant number of cases, chronic pulmonary conditions ordissemination to the meninges, bones, and joints can result, leading toacute, life-threatening disease. Migrant laborers who are exposed toCoccidioides spp. are a highly mobile and underrepresented population,and unfortunately, this disease is under-reported and receivesinsufficient attention from the general medical community. A variety ofapproaches have been used to fight coccidioidomycosis, including soiltreatments, but only a vaccine can completely eliminate this “emergingdisease.”

Cellular Defenses Against Coccidioides spp.

Polymorphonuclear leukocytes (PMNs) are only able to partially inhibitgrowth of the pathogen, and are unable to kill the organism. PMNs areslightly more effective in inhibiting growth of arthroconidia thanmature spherules. Since mature fungal spherules are typically 40-120 μmin diameter, a single PMN is unable to phagocytose the fungal cell.Endospores, on the other hand, are more sensitive to growth inhibitionby these host cells. Most investigators of cellular immune response toCoccidioides spp. believe that macrophages are the pivotal effectorcells in coccidioidomycosis. This concept has arisen from the generalparadigm for granulomatous diseases: activated T-lymphocytes secretecytokines, which activate macrophages, inducing the formation of agranuloma, which kills or contains the organism. As the spheruledevelops and matures, it becomes more resistant to macrophages, so thatless than 10% of mature spherules are killed. It has been suggested thatspecific immunologic suppression elicited by Coccidioides spp. antigensprevents an effective T-cell response.

Cryptococcosis

In certain embodiments of the present invention, methods, compositionsand treatments for a fungal organism infection can concern treatment foror prevention of a Cryptococcosis-associated condition. C. neoformanscan be an intracellular as well as an extracellular pathogen. It iseasily found extracellularly in cerebral spinal fluid and lung tissue.It can also be localized inside macrophages and neutrophils where it hasbeen shown to replicate.

Antibody and cell-mediated responses can provide important protectioneven against an intracellular pathogen. The mammalian host has severaldefenses against C. neoformans that include components from innate,humoral, and cell-mediated immunity. Subjects are continually exposed toC. neoformans from the environment, a powerful innate immunityrepresents part of the protection afforded the normal host. This appearsto be inherent in normal macrophage, monocyte, and neutrophil function.

Vaccination against cryptococcosis presents the innovative idea of notonly aiming to protect an immunocompromised population, but alsopossibly expecting an immune response in that population. In oneembodiment, a vaccine against cryptococcosis is contemplated forexample, to treat a non-immunosuppressed or immunosuppressed subject. Inone particular example, immunosuppressed patients such as HIV patientsare contemplated. In accordance with this embodiment, an HIV patient maybe vaccinated immediately after diagnosis, but before their CD4⁺ T cellpopulation decreases.

In other embodiments, any of the vaccines detailed herein may bedirected towards other fungal organisms, for example, Coccidioides andAspergillus. It is contemplated herein that any fungal pathogendisclosed may have one or more virulence factors which may includesurface proteins, cell-wall carbohydrates, secreted factors, anchoredsurface molecules, modes of action to invade a host, etc. Any virulencefactor associated with a fungal pathogen may be used herein as apotential target to transform yeast or other fungi of the presentinvention to generate a vaccine against that fungal pathogen.

In certain embodiments of the present invention, the fungal cellscomprise transgenic cells, non-transgenic cells or a combinationthereof. In other embodiments, the fungal cells can include, but are notlimited to, Saccharomyces spp., Aspergillus spp., Cryptococcus spp.,Coccidioides spp., Neurospora spp., Histoplasma spp., Blastomyces spp.,and a combination thereof.

In certain particular embodiments, Saccharomyces spp. cells can bespores, vegetative cells, germlings, or a combination thereof. In otherembodiments, the Neurospora spp. cells can be vegetative hyphae, aerialhyphae, macroconidia, germinating macroconidia, microconidia,germinating microconidia, ascospores, germinating ascospores, or acombination thereof.

Recombinant Yeast as an Antigen Delivery System

In one example, a yeast vaccine formulation directly accesses DCs, thecritical immune responsive cells of the body. In order for DCs topresent antigens efficiently to naive T cells, immature DCs must beactivated to mature, as evidenced by the up-regulation of MHC andco-stimulatory molecules. Mature DCs are then capable of prolongedantigen presentation and the production of cytokines, such as IL-12,that are critical for the induction of cellular immune responses. Uptakeof yeast by DCs increased surface expression of the co-stimulatorymolecules CD40, CD80 and CD86, MHC class II, and the adhesion moleculeICAM, to levels comparable to that induced by exposure to bacteriallipopolysaccharide (LPS), a potent DC maturation factor. As furtherevidence of yeast-induced activation, DCs incubated with yeast producedsignificant amounts of IL-12 that rivalled levels induced by exposure toLPS. These results show that yeast trigger DC maturation, responses thatwould be essential for vaccine-induced immunity.

It has been shown that certain yeast vaccine technology can beformulated with a variety of antigens, including 5 from HIV, 3 fromHepatitis B, 1 from Hepatitis C, and 2 from lung cancer cells and, ineach case, the yeast-expressed antigen effectively stimulated protectivecell-mediated immunity. In addition, yeast as a vaccine vehicle providesan adjuvant effect, such that dendritic cells are triggered to directlytake up yeast, causing the DCs to mature and to present theyeast-associated antigens to the host immune system. Further, recentresults have shown that heat-killed yeast cells expressing putativeantigens are as immunogenic and protective as live yeast cellsexpressing putative antigens. In addition, yeast cells expressingantigens elicited Th1 responses. Therefore, recombinant-yeast vaccineformulations may elicit systemic, antigen-specific, CD4 and CD8-basedprotective immunity. Also, DCs have been shown to internalize yeast andpresent recombinant antigens to naive class I and class IIMHC-restricted T cells. It has been shown that yeast exhibit adjuvantactivity for DC maturation and immune signalling. Yeast might beproviding an adjuvant effect to promote immune responses to theyeast-associated antigens. These results show that i) yeast uptaketriggers DC maturation; ii) yeast-expressed recombinant antigens areefficiently presented by MHC class I and MHC class II pathways of DCs,and, iii) yeast provides an adjuvant effect to enhance DC-stimulation ofnaive T cells.

In another example, a CD8-dependent protective immunity has beenelicited in vaccinated mice. For example, EL-4 CD4⁺ T lymphoma cells(H-2^(b)) transfected with cDNA encoding chicken ovalbumin (E.G7-OVA)and ovalbumin-expressing melanoma (B16-OVA) mouse tumor models have beenemployed to test vaccine candidates that induce protective CTL response.Mice vaccinated with OVAX, but not mock-vaccinated animals, wereprotected from E.G7-OVA or B16-OVA tumor formation. Protective immunitywas CD8-dependent, since OVAX-vaccinated animals were not protected fromtumor challenge in CD8^(−/−) knockout mice.

Kits

In still further embodiments, the present invention concerns kits forthe methods described herein. In one embodiment, a fungal organismtreatment or (such as a pathogenic or non-pathogenic fungus) preventionkit is contemplated. In another embodiment, a kit for prophylactictreatment of a subject having or suspected of developing a fungalinfection is contemplated. In a more particular embodiment, a kit forprevention or treatment of a subject having or suspected of developing afungal-induced infection is contemplated. In accordance with thisembodiment, the kit may be used to treat or vaccinate a subject for oragainst a fungal infection.

The kits may include a composition including at least a portion of anon-viable fungus within a tube, a vial or other suitable vessel. Inaddition, the kit may include one or more dose(s) of the composition foradministration to a subject having or exposed to a fungal organisminfection by a healthcare provider. In another embodiment, the kit maybe a portable kit for use at a specified location outside of ahealthcare facility.

The container means of any of the kits will generally include at leastone vial, test tube, flask, bottle, syringe or other container means,into which the testing agent, may be preferably and/or suitablyaliquoted.

Nucleic Acids

In various embodiments, isolated nucleic acids may be used to modify ortransform a fungal organism contemplated in the present invention. Theisolated nucleic acid may be derived from genomic DNA, RNA orcomplementary DNA (cDNA). In other embodiments, isolated nucleic acids,such as chemically or enzymatically synthesized DNA, may be of use forgeneration of oligonucleotides for transformation of a fungal organism.

A “nucleic acid” includes single-stranded and double-stranded molecules,as well as DNA, RNA, chemically modified nucleic acids and nucleic acidanalogs. It is contemplated that a nucleic acid may be of 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, about 110, about 120, about 130, about 140, about 150,about 160, about 170, about 180, about 190, about 200, about 210, about220, about 230, about 240, about 250, about 275, about 300, about 325,about 350, about 375, about 400, about 425, about 450, about 475, about500, about 525, about 550, about 575, about 600, about 625, about 650,about 675, about 700, about 725, about 750, about 775, about 800, about825, about 850, about 875, about 900, about 925, about 950, about 975,about 1000, about 1100, about 1200, about 1300, about 1400, about 1500,about 1750, about 2000 or greater nucleotide residues in length, up to afull length protein encoding or regulatory genetic element.

Construction of Nucleic Acids

Isolated nucleic acids may be made by any method known in the art, forexample using standard recombinant methods, synthetic techniques, orcombinations thereof. In some embodiments, the nucleic acids may becloned, amplified, or otherwise constructed.

Nucleic acids of use in the present invention may includeoligonucleotides that include at least a portion of sequences from avirulence factor (e.g., including but not limited to, antigen-2/prolinerich antigen, hemolysin, aspf2, dpp5, dpp4, chitin deacetylase,catalase), a disease-associated factor (e.g., tumor associated antigens,cytokines, viral-associated antigens, bacterial-associated antigens) orother peptides or proteins. In another example, a multi-cloning sitecomprising one or more endonuclease restriction sites may be added. Anucleic acid may be attached to a vector, adapter, or linker for cloningof a nucleic acid. Additional sequences may be added to such cloning andsequences to optimize their function, to aid in isolation of the nucleicacid, or to improve the introduction of the nucleic acid into a cell.Use of cloning vectors, expression vectors, adapters, and linkers iswell known in the art.

Recombinant Methods for Constructing Nucleic Acids

Isolated nucleic acids may be obtained from fungal, bacterial, viral orother sources using any number of cloning methodologies known in theart. In some embodiments, oligonucleotide probes which selectivelyhybridize, under stringent conditions, to the nucleic acids are used toidentify a sequence of interest. Methods for construction of nucleicacid libraries are known and any such known methods may be used. (See,e.g., Current Protocols in Molecular Biology, Ausubel, et al., Eds.,Greene Publishing and Wiley-Interscience, New York (1995); Sambrook, etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Vols. 1-3 (1989); Methods in Enzymology, Vol. 152, Guide toMolecular Cloning Techniques, Berger and Kimmel, Eds., San Diego:Academic Press, Inc. (1987)).

Nucleic Acid Screening and Isolation

Genomic DNA, RNA or cDNA may be screened for the presence of anidentified genetic element of interest using a probe based upon one ormore sequences. Various degrees of stringency of hybridization may beemployed in the assay. As the conditions for hybridization become morestringent, there must be a greater degree of complementarity between theprobe and the target for duplex formation to occur. The degree ofstringency may be controlled by temperature, ionic strength, pH and/orthe presence of a partially denaturing solvent such as formamide. Forexample, the stringency of hybridization is conveniently varied bychanging the concentration of formamide within the range of 0% to 50%.The degree of complementarity (sequence identity) required fordetectable binding will vary in accordance with the stringency of thehybridization medium and/or wash medium.

High stringency conditions for nucleic acid hybridization are well knownin the art. For example, conditions may comprise low salt and/or hightemperature conditions, such as provided by about 0.02 M to about 0.15 MNaCl at temperatures of about 50° C. to about 70° C. Other exemplaryconditions are disclosed in the following Examples. It is understoodthat the temperature and ionic strength of a desired stringency aredetermined in part by the length of the particular nucleic acid(s), thelength and nucleotide content of the target sequence(s), the chargecomposition of the nucleic acid(s), and to the presence or concentrationof formamide, tetramethylammonium chloride or other solvent(s) in ahybridization mixture. Nucleic acids may be completely complementary toa target sequence or may exhibit one or more mismatches.

Nucleic Acid Amplification

Nucleic acids of interest may also be amplified using a variety of knownamplification techniques. For instance, polymerase chain reaction (PCR)technology may be used to amplify target sequences directly from RNA orcDNA. PCR and other in vitro amplification methods may also be useful,for example, to clone nucleic acid sequences, to make nucleic acids touse as probes for detecting the presence of a target nucleic acid insamples, for nucleic acid sequencing, or for other purposes. Examples oftechniques of use for nucleic acid amplification are found in Berger,Sambrook, and Ausubel, as well as Mullis et al., U.S. Pat. No. 4,683,202(1987); and, PCR Protocols A Guide to Methods and Applications, Innis etal., Eds., Academic Press Inc., San Diego, Calif. (1990). PCR-basedscreening methods have been disclosed. (See, e.g., Wilfinger et al.BioTechniques, 22(3): 481-486 (1997))

Synthetic Methods for Constructing Nucleic Acids

Isolated nucleic acids may be prepared by direct chemical synthesis bymethods such as the phosphotriester method of Narang et al., Meth.Enzymol. 68:90-99 (1979); the phosphodiester method of Brown et al.,Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramidite method ofBeaucage et al., Tetra. Lett. 22:859-1862 (1981); the solid phasephosphoramidite triester method of Beaucage and Caruthers, Tetra. Letts.22(20):1859-1862 (1981), using an automated synthesizer as inNeedham-VanDevanter et al., Nucleic Acids Res., 12:6159-6168 (1984); orby the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis generally produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. While chemical synthesis of DNA is bestemployed for sequences of about 100 bases or less, longer sequences maybe obtained by the ligation of shorter sequences.

Covalent Modification of Nucleic Acids

A variety of cross-linking agents, alkylating agents and radicalgenerating species may be used to bind, label, detect, and/or cleavenucleic acids. For example, Vlassov, V. V., et al., Nucleic Acids Res(1986) 14:4065-4076, disclose covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervanalso showed sequence-specific cleavage of single-stranded DNA mediatedby incorporation of a modified nucleotide which was capable ofactivating cleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, R. B.,et al., J Am Chem Soc (1989) 111:8517-8519 disclose covalentcrosslinking to a target nucleotide using an alkylating agentcomplementary to the single-stranded target nucleotide sequence. Aphotoactivated crosslinking to single-stranded oligonucleotides mediatedby psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988)27:3197-3203. Use of crosslinking in triple-helix forming probes wasalso disclosed by Home, et al., J Am Chem Soc (1990) 112:2435-2437. Useof N4, N4-ethanocytosine as an alkylating agent to crosslink tosingle-stranded oligonucleotides has also been disclosed by Webb andMatteucci, J Am Chem Soc (1986) 108:2764-2765; Nucleic Acids Res (1986)14:7661-7674; Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991).Various compounds to bind, detect, label, and/or cleave nucleic acidsare known in the art. See, for example, U.S. Pat. Nos. 5,543,507;5,672,593; 5,484,908; 5,256,648; and, 5,681,941.

Nucleic Acid Labeling

In various embodiments, tag nucleic acids may be used to trace oridentify a particular nucleic acid sequence contemplated herein. Anumber of different labels may be used, such as fluorophores,chromophores, radio-isotopes, enzymatic tags, antibodies,chemiluminescent, electroluminescent, affinity labels, etc. One of skillin the art will recognize that these and other label moieties notmentioned herein can be used. Examples of enzymatic tags include urease,alkaline phosphatase or peroxidase. Colorimetric indicator substratescan be employed with such enzymes to provide a detection means visibleto the human eye or spectrophotometrically. A well-known example of achemiluminescent label is the luciferin/luciferase combination.

In certain embodiments, the label may be a fluorescent, phosphorescentor chemiluminescent label. Exemplary photodetectable labels may beselected from the group consisting of Alexa 350, Alexa 430, AMCA,aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy5, 6-FAM, dansylchloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole),Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue,phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet,cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid,erythrosine, phthalocyanines, azomethines, cyanines, xanthines,succinylfluoresceins, rare earth metal cryptates, europiumtrisbipyridine diamine, a europium cryptate or chelate, diamine,dicyanins, La Jolla blue dye, allopycocyanin, allococyanin B,phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin,phycoerythrin R, REG, Rhodamine Green, rhodamine isothiocyanate,Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol),Tetramethylrhodamine, and Texas Red. These and other labels areavailable from commercial sources, such as Molecular Probes (Eugene,Oreg.).

In certain embodiments, it is contemplated that a vaccine will beadministered to a subject for vaccination against and/or treatment of afungal infection. It is also contemplated herein that any of thevaccines disclosed may be administered to a subject having or suspectedof developing a condition due to exposure or potential exposure to afungal pathogen. In one embodiment, a subject may include a human. Inother embodiments a subject may include other mammals, birds, andreptiles. Any of the methods disclosed herein may be used in combinationwith other anti-fungal agents or therapies to treat a subject in orderto achieve the desired results.

In another embodiment, it is contemplated that a subject can bevaccinated and/or treated using a composition against a fungal pathogendisclosed herein before infection, during infection, after infection orcombination therefore of. In one particular embodiment, a compositiondisclosed herein may be used to vaccinate a subject against a particularfungal organism to reduce the risk of a fungal infection in the subject.In another embodiment, a composition disclosed herein may be used tovaccinate a subject against one or more fungal organism to reduce therisk of a fungal infection in the subject. In certain embodiments, avaccine containing non-viable fungal cells of one fungal type whenadministered to a subject may reduce the onset or progression of fungalinfection of another fungal type in the subject.

Exemplary Dosing: In one embodiment, a dose may consist of around 1×10⁷yeast cells to 20×10⁷ yeast cells per dose. And may be given beforeinfection, during infection, after infection or a combination thereforeof. The dose may be given daily, every other day, biweekly, weekly,seasonally or yearly until a desired effect is achieved.

A composition of the present invention may be administered to a subjectin an appropriate carrier or diluent or pharmaceutically acceptablecarrier, co-administered with enzyme inhibitors or in an appropriatecarrier such as liposomes. The term “pharmaceutically acceptablecarrier” as used herein is intended to include diluents such as salineand aqueous buffer solutions. To administer a compound that stimulatesan immune response by other than parenteral administration, it may benecessary to coat the compound with, or co-administer the compound with,a material to prevent its inactivation. Enzyme inhibitors includepancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) andtrasylol. Liposomes include water-in-oil-in-water emulsions as well asconventional liposomes. The active agent may also be administeredparenterally or intraperitoneally. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations maycontain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria. The pharmaceutically acceptable carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of microorganisms can beachieved by various antibacterial and antifungal agents (i.e., parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. A compound such as aluminum monostearate and gelatin can beincluded to prolong absorption of the injectable compositions.

Sterile injectable solutions can be prepared by incorporating activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains adispersion medium and other required ingredients from those enumeratedabove. In the case of sterile powders for the preparation of sterileinjectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active ingredient(i.e., a chemical agent, antibody, etc.) plus any additional desiredingredient from a previously sterile-filtered solution thereof.

When the active agent is suitably protected, as described above, thecomposition may be orally administered (or otherwise indicated), forexample, with an inert diluent or an assimilable edible carrier. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of compound such as non-viablefungal cells calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

If needed for a particular use, the biological material can beextensively dialyzed to remove undesired small molecular weightmolecules and/or lyophilized for more ready formulation into a desiredvehicle, where appropriate. The active compounds will then generally beformulated for parenteral administration (i.e., formulated for injectionvia the intravenous, intramuscular, sub-cutaneous, intralesional, oreven intraperitoneal routes). The preparation of an aqueous compositionthat contains an active component or ingredient will be known.Typically, such compositions can be prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for use inpreparing solutions or suspensions upon the addition of a liquid priorto injection can also be prepared; and the preparations can also beemulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid. Itmust be stable under the conditions of manufacture and storage and mustbe preserved against the contaminating action of microorganisms, such asbacteria and fungi.

Solutions of the active compounds as free-base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. Prolonged absorption of the injectable compositions canbe brought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The preparation of more, or highly,concentrated solutions for direct injection is also contemplated, wherethe use of DMSO as solvent is envisioned to result in extremely rapidpenetration, delivering high concentrations of the active agents to asmall area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g., liposomal formulations; time-releasecapsules; and any other form currently used.

In another embodiment, nasal solutions or sprays, aerosols or inhalantsmay be used to deliver the compound of interest. Nasal solutions areusually aqueous solutions designed to be administered to the nasalpassages in drops or sprays. Nasal solutions are prepared so that theyare similar in many respects to nasal secretions. Thus, the aqueousnasal solutions usually are isotonic and slightly buffered to maintain apH of 5.5 to 6.5. In addition, antimicrobial preservatives, similar tothose used in ophthalmic preparations, and appropriate drug stabilizers,if required, may be included in the formulation. Various commercialnasal preparations are known and include, for example, antibiotics andantihistamines and are used for asthma prophylaxis Inhalationpreparations may include solutions or dry powder formulations that arecommonly used along with a propellant in the formulation of therapeuticsused for the treatment of asthmatics.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders. Incertain defined embodiments, oral pharmaceutical compositions willcomprise an inert diluent or assimilable edible carrier, or they may beenclosed in hard or soft shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 75% of theweight of the unit, or preferably between 25-60%. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

Particularly preferred are methods in which the therapeutic compound(s)are directly administered as a pressurized aerosol or nebulizedformulation to the patient's lungs via inhalation. Such formulations maycontain any of a variety of known aerosol propellants useful forendopulmonary and/or intranasal inhalation administration. In addition,water may be present, with or without any of a variety of cosolvents,surfactants, stabilizers (e.g., antioxidants, chelating agents, inertgases and buffers).

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Methods

Proteins to be tested Each protein from a fungal pathogen was chosenbecause of its identification as a virulence factor (e.g., strainslacking the protein were less virulent that the wild type), as animmunogen (infected animals generated antibodies to the candidateprotein), and/or was found on the cell surface of conidia or hyphae. Inaddition, none of the proteins had cognates with significant (<35%)homology to mammals and to yeast.

Exemplary isolation of DNA containing the open reading frame encodingeach protein: The Open Reading Frame (ORF) of each gene was obtained byPCR using gene specific primers and in certain embodiments, a highquality cDNA library as template. In other embodiments, the ORF ofcandidate genes was obtained using plasmid DNA containing the gene ofinterest as a template for amplification by PCR. Each PCR product wasgel purified and ligated into the TA cloning vector, pCR2.1, using thepCR2.1 TOPO kit from InVitrogen (Carlsbad, Calif.). Resulting plasmidDNAs were used to transform competent E. coli cells and plasmid DNAisolated from midi-preps using methods standard to those of skill in theart.

In certain embodiments the gene specific PCR primers encoded a c-mycepitope such that the c-myc epitope would be generated in the protein atthe carboxy terminus.

In certain methods, each ORF was excised from each plasmid DNA using therestriction endonuclease EcoR1 and ligated into the EcoR1 site ofpYEX-BX. Each resulting ligated plasmid was used to transform competentE. coli cells and plasmid DNA isolated from midi-preps. Each ORF DNA wassequenced to ensure that it is in frame with the copper-induciblepromoter and no PCR errors were present.

In one exemplary method an empty vector control was generated. As oneexemplary control, an empty vector control was generated a pYEX-BX(Clonetech) vector containing the DNA encoding a c-myc epitope butlacking a fungal gene of interest. A 51 base pair template for PCR wascreated that contained a methionine, the sequence that encodes the c-mycprotein and two stop codons. The PCR primers also included a 5′ BamH Isite and a 3′ EcoR I restriction site that could be used to excise thetag from the pCR 2.1 vector and inserted into the pYEX-BX vector(Clonetech). The DNA and amino acid sequences are shown in SEQ ID NOs:1and 2:

atg gaa cag aag ttg att tcc gaa gaa gac ctc gag Met Glu Gln Lys Leu IleSer Glu Glu Asp Leu Glu c-myc control sequence

This template was amplified by PCR under the following conditions: 25 μLreactions were performed in PCR Ready Bead tubes containing 500 ng ofcDNA library, 2 μM primers, and 13 μL sterile water. Reactions wereperformed using a Perkin Elmer 2400 Thermocycler under the followingconditions: an initial cycle at 94° C. for 5 min, 30 cycles at 94° C.for 1 min, 50° C. for 1 min, 72° C. for 1 min, and a final extension at72° C. for 7 min.

The PCR reactions were separated by agarose gel electrophoresis using a3% (w/v) agarose gel. The 51 by band was excised from the gel, purifiedand ligated into pCR 2.1 (Invitrogen). The ligated vector was used totransform competent E. coli cells and vector DNA was subsequentlyisolated using standard techniques. DNA isolated from several isolateswas used as template for PCR reactions to confirm the presence of thec-myc insert.

Several isolates were grown, cells harvested, plasmid DNA isolated andthe DNA sequence of the c-myc were determined. A comparison the sequenceobtained empirically with the published sequence revealed that no PCRerrors were apparent. An ATG was added by the PCR primers to ensure thetranslation of the c-myc peptide.

Transformation of Fungal Cells Several exemplary fungal cells that canbe transformed to express vaccine proteins can include, but are notlimited to, Saccharomyces spp., Aspergillus spp., Cryptococcus spp.,Coccidioides spp., Neurospora spp., Histoplasma spp., Blastomyces spp.,and a combination thereof.

Exemplary method of transformation of S. cerevisiae. After isolatingeach of the genes of interest and forming each yeast expression vector(as described), DNA vectors to individually transform yeast cells wereused. The transformation protocol was as follows: 50 μL of YPD broth wasinoculated with W303-1B Saccharomyces cerevisiae and the culture grownovernight at 30° C. with shaking. The desired OD600 for transformationwas between 0.1-0.6 (˜2×107 c/mL). Cells were harvested bycentrifugation at 2000×g for 10 minutes, the pellet was washed in 25 mLsterile water and cells were again centrifuged at 2000×g for 10 minutes.The pellet was resuspended in 1 mL 0.1M lithium acetate (LiAC),centrifuged at 1000×g for 30 seconds and the supernatant removed. Thispellet was again resuspended in 400 μL 0.1M LiAC and the cell suspensionmixed until the cells were completely suspended. Fifty milliliters ofthis cell suspension was used for each transformation. The cells werecentrifuged at 3000×g for 30 seconds and the supernatant discarded. Tothe cells was added 240 μL 50% (w/v) PEG, 36 μL 1M LiAC, 25 μL denaturedcarrier DNA, and 50 μL of previously diluted water containing plasmidDNA (0.1-10 μg). Cells were mixed until the pellet completelyresuspended, mixtures were incubated at 30° C. for 30 minutes, and heatshocked at 42° C. for 20-25 min. The cells were harvested bycentrifugation at 3000×g for 30 seconds, gently resuspended in 1 mL ofwater and spread onto Yeast Nitrogen Base medium (YNB) (e.g., Difco)minus uracil plates. The plates were incubated at 30° C. for 3-4 days.

Identification of yeast cells expressing candidate proteins In oneexample, a single yeast colony was obtained from YNB minus uracil platesand grown for overnight at 30° C. with shaking in 250 mL flaskscontaining 50 mL YNB minus uracil medium. Each culture was diluted to afinal concentration of 0.2 (OD600) in 50 mL YNB minus uracil interimcultures. The cultures were grown at 30° C. until the OD600 had doubled(approximately 5-6 hours). The OD600 of the interim cultures wasdetermined a final time before the cultures were diluted to a finalconcentration of 0.02 OD600 units in 50 mL of YNB minus uracil. Thesecultures were grown for 1 hour with shaking at 30° C. before 0.4 mMCuSO₄ (final concentration) was added. As a control, to a duplicateculture, 0.4 mM NaSO₄ was added to show that the protein is not producedin the absence of copper. Once the CuSO₄ and NaSO₄ were added, thecultures were grown overnight (18 hours) at 30° C. with shaking (FIG.1).

Induced and uninduced cultures were harvested after overnight incubationby centrifugation at 550×g for 10 minutes. Each cell pellet was washedwith 25 mL ice cold water to remove any residual medium and the cellswere harvested again by centrifugation. Each pellet was then resuspendedin 1 mL of ice cold water and divided into two 1.5 mL eppendorf tubesfor centrifugation. After the removal of the supernatant, one tube wasfrozen at −80° C. for storage and the other was lysed for proteindetermination. Cells were lysed in the following manner: 100 μL of glassbeads were added followed by the addition of 200 μL 2× sample lysisbuffer (0.1M Tris, pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS, 0.5% (v/v)(β-mercaptoethanol). Tubes were mixed by vortexing for 1 min toresuspend and lyse the cells and mixtures were boiled for 3 min todenature the proteins for SDS-PAGE gel electrophoresis. Debris wasremoved by centrifugation at 550×g for 5 min. and the cleared celllysates were transferred to new tubes.

Cell lysates were loaded into the wells of 15% Tris-HCl SDS-PAGE gels(example: Bio Rad, Hercules, Calif.) in 15 μL aliquots and separated byelectrophoresis at 60-80 V for 2-3 hours. Proteins were then transferredfrom the gel to a PVDF [poly (vinylidine fluoride)] membrane using amini-genie transfer apparatus (Idea Scientific, Minneapolis, Minn.) for1 hour at 12 volts.

PVDF membranes were blocked in TBST (100 mM Tris, 1.5 M NaCl and 0.1%[v/v] Tween 20) containing 5% (w/v) powdered milk (Carnation) for 1 hourat room temperature. Blocked membranes were then incubated with primaryanti-c-myc epitope antibody (1:1000 dilution) in TBST containing 1% milkovernight at 4° C. The next morning the membranes were washed five timesfor 15 minutes each in TBST before secondary antibody was added.Secondary antibody conjugated to horse radish peroxidase (HRP) was addedin TBST containing 1% (w/v) milk and incubated for 1 hour at roomtemperature. The membrane was washed five times for 5 minutes each inTBST before exposure to ECL reagents. The membrane was exposed to ECL(Pierce Chemical Company, Rockford, Ill.) for 1 minute then developedusing an Alpha-Innotech (San Leandro, Calif.) digital system.

Determination of Optimal Growth Conditions of Yeast Expressing CandidateProteins

Initial 50 mL cultures were started with 8004 of each glycerol stockthat was frozen at a concentration of 1×10⁸ cells/mL. Cultures weregrown overnight at 30° C. with shaking. The next morning the cultureswere counted using a hemocytometer as well as for the OD600determination using a SpectraMax 340 plate reader (Molecular Devices,Sunnyvale, Calif.). Each culture was then diluted to OD600 of 0.1, 0.2,or 0.3. Duplicate cultures were grown in 250 mL Erlenmeyer flasks (50 mLcultures) as well as in 300 mL Klett flasks (containing 60 mL ofmedium). Samples were taken every hour from the 250 mL Erlenmeyer flasksfor cell counting and OD600 readings, while Klett readings were takenusing a Klett-Summerson Photoelectric colorimeter (Klett-Summerson,N.Y.). By using these methods, not only could the growth curve becharacterized, but the OD600 could be correlated, cell counts, and Klettreadings thereby allowing use of any of the three methods in futureexperiments. The following correlation was determined; 50 Klettunits=0.25 OD600 units=2×10⁷ c/mL. From these data, the doubling time ofthe culture was determined to be approximately 5 hours. An exemplarygrowth curve for one such transformant is shown in FIG. 2.

Once the growth curves at each of the three initial inoculationconcentrations were determined, the new variable of various CuSO₄concentrations was added. Copper sulfate was tested at the followingconcentrations: 0, 0.2, 0.3, and 0.4 mM. The protocol to induce pYEX-BXsuggested a CuSO₄ range of 0.2-0.5 mM and it had previously beendetermined that 0.5 mM was potentially toxic to the yeast. As in theexperiment above, duplicate cultures were inoculated in 250 mLErlenmeyer flasks and 300 mL Klett flasks. Samples were taken every hourfrom the 250 mL Erlenmeyer flasks for cell counting and OD600 readings,while Klett readings were taken from the Klett flasks. The lowerinoculum grew the best upon the addition of CuSO₄. The correlationbetween Klett units, OD600 units, and cell counts was the same asdetermined previously. The doubling time of the each culture was again 5hours and was independent of copper concentration. The growth curve withcopper for one such transformant at each innoculumis shown in FIG. 1.

Preparation of Vaccine

In one exemplary method, overnight cultures were started by adding 1 mLof a glycerol stock of yeast cells to 50 mL of YNB minus uracil mediumin a 250 mL flask. Cultures were grown overnight at 30° C. with shaking.The next morning the OD600 was determined and a 50 mL interim culturewas inoculated at a final OD600 of 0.2 and grown at 30° C. with shakinguntil it had doubled (4-5 hours). The optical density was subsequentlydetermined for the interim culture which was used to inoculate a final250 mL culture at 0.02 (final OD600) and grown for 1 hour at 30° C. withshaking. After the hour of growth, 0.4 mM CuSO₄ (final concentration)was added to induce the production of each candidate protein. Thisinduced culture was grown for 16 to 18 hours at 30° C. with shaking.Because the doubling time for copper-induced cells was approximately 5hours, these induced cultures were in mid to late log after about 18hours of growth.

Heat-Killed Preparations

In one example, cells from cultures were harvested by centrifugation at1900 g for 5 min at 4° C. Cultures were washed 4 times with 100 mL roomtemperature PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH2PO₄) toremove any residual medium and copper from the cells. Cells wereresuspended in 3 mL room temperature PBS and then added to 200 mL PBSthat had been pre-warmed to 56° C. The cells were subsequently heatkilled by incubation at 56° C. in a water bath for 1 hour, withinversion of tubes every 10 min. to ensure that all cells were heatkilled. Cells were harvested by centrifugation (1900×g for 5 min at 4°C.) and then washed 4 times with 100 mL with room temperature PBS. Cellswere counted and resuspended at a final concentration of 4×108 c/mL inPBS, aliquoted in 200 mL aliquots and stored at 4° C.

Yeast cells can be heat-killed by incubation in sterile, pyrogen-freesaline at 56° C. for 1 hr, washed in saline, resuspended at 2×10⁸cells/mL in sterile saline, and stored at 4° C. Each batch is expectedto yield ˜2×10¹⁰ yeast cells, of which a vaccine dose for the animalstudies will consist of ˜1×10⁷ to 20×10⁷ cells.

Test for Live Cells in Heat-Killed Preparations

In one method, to ensure that the heat treatment has inactivated >99.99%of the yeast cells, 1×10⁷ heat-killed cells were incubated onagar-solidified rich medium for three days at 30° C. and the number ofresulting yeast colonies (if any) determined. Only batches of cells withsurvival rates of <0.01% are to be used. To confirm that no livemicroorganisms, e.g., bacteria, were present, fluid thioglycolate medium(as described in the USP 24 NF19) may be inoculated with each batch ofheat-killed yeast cells. The test mixtures were incubated for 14 days at32° C. and examined visually for growth. Only batches of heat-killedcells in which minimal to no microbial growth was observed were used.

Endotoxin Levels of Each Batch of Vaccine

Bacterial cell-wall fragments and other pyrogens can often obscureimmune responses. Pyrogen-free containers, liquids, and media were usedto minimize spurious endotoxin contamination. Recently, the FDA hasallowed use of a test for bacterial endotoxin levels to substitute forrabbit pyrogenicity tests. The endotoxin levels of each vaccineformulation were quantitated using a G test (Fungitec, Seikagaku Corp.,Tokyo, Japan) under the U.S. Pharmacopeia (USP) guidelines. This testrelies on the factor G component of horseshoe crab amoebocyte lysate.Unfortunately, this component is sensitive to (1,3)β-glucan whilefactors B and C are sensitive to endotoxin. Since it is expected that asmall amount of fungal cell-wall glucan is present in heat-killed yeastpreparations and these may lead to false positives, all samples prior totest were treated with (1,3)β-glucanase, which will destroy any(1,3)β-glucan. This procedure thus will provide an accuratedetermination of the endotoxin levels present. Only batches ofheat-killed cells that contain <0.5 EU/mL were used.

Example 1

In certain exemplary methods, the fungal organism Aspergillus wasexamined.

Efficacy of four A. fumigatus antigens protecting mice from systemicaspergillosis In one exemplary method, the protective efficacy of 4 A.fumigatus antigens expressed in Saccharomyces sp. in an experimentalmodel of systemic murine aspergillosis was tested. Efficacy wasevaluated in terms of survival and tissue burden. See FIG. 3 as oneexemplary strategy for vaccine generation and animal testing.

Antigens In this example, vaccines preparations containing 6×10⁷heat-killed yeast cells/150 microliters were administered s.c. using twoinjection sites (0.075 ml each) on days 28, 21 and 14 before infection.Groups pretreated with Myc (yeast containing and expressing only thec-myc tag) or PBS were used as controls.

Animals 60 CD-1 male mice weighting 30 g approx. were used.

Inoculum A total of 1.3×10⁷ conidia of A. fumigatus, strain AF-10, wereinoculated via lateral tail vein into the CD-1 mice.

In this exemplary method, 16 days after infection, animals wereeuthanatized by CO₂ anoxia and kidney and brain were removed. Each organwas homogenized, diluted and plated on SDA plates for CFU determination.

Survival

Comparisons showed that animals immunized with Hemolysin (Hemo or HEMO)antigen had an increased survival time in comparison with the PBScontrol group (p=0.012). In addition, the MYC control treated group didhave an increased survival time in comparison with the PBS control groupalthough this did not reach statistical significance (p=0.089) (FIG. 4).

In another example, the protective effect of the A. fumigatus antigen(HEMO) expressed in Saccharomyces sp. in a systemic murine model ofaspergillosis was evaluated. The animal model showed low mortality inthe control groups i.e., PBS and MYC, and HEMO pretreatment did notsignificantly prolonged the survival of the animals. However, the tissueburden study demonstrated that animals receiving three doses of the HEMOantigen before the infection had significantly lower CFU of the fungusin the kidneys and brain in comparison with the PBS-treated controlanimals (FIG. 12). These results demonstrate that HEMO pretreatmentprovides protective resistance against systemic aspergillosis.

Exemplary Cloning of AspF 2. Published DNA and predicted amino acidsequences to design PCR primers to amplify the ORF from the Aspergillusfumigatus cDNA library were used. The DNA and amino acid sequences ofAspF 2 are presented in diagram form in SEQ ID NOs:3 and 4. Thepositions of the 5′ and 3′ primers use to obtain the predicted PCRproduct are also shown in SEQ ID NOs:3 and 4.

atg ctt gtg gcc acc ctc cct acc tcc ccc gtc ccc atc gcg gcg cga  48 MetLeu Val Ala Thr Leu Pro Thr Ser Pro Val Pro Ile Ala Ala Arg1               5                   10                  15 gca acc ccccac gaa ccc gtc ttc ttc tcc tgg gac gct ggc gcg gtg  96 Ala Thr Pro HisGlu Pro Val Phe Phe Ser Trp Asp Ala Gly Ala Val            20                  25                  30 acc tcg ttc cccatc cac tcc agc tgc aat gcg acc cag cgc cgg cag 144 Thr Ser Phe Pro IleHis Ser Ser Cys Asn Ala Thr Gln Arg Arg Gln        35                  40                  45 atc gag gcc ggc ctgaac gag gcg gtc gag ctc gcc cgg cac gcc aag 192 Ile Glu Ala Gly Leu AsnGlu Ala Val Glu Leu Ala Arg His Ala Lys    50                  55                  60 gcc cac atc ctc cgc tggggc aac gag agc gag atc tac cgg aag tac 240 Ala His Ile Leu Arg Trp GlyAsn Glu Ser Glu Ile Tyr Arg Lys Tyr65                  70                  75                  80 ttt ggcaac cgg ccc acc atg gag gcc gtc ggt gcc tac gat gtc atc 288 Phe Gly AsnArg Pro Thr Met Glu Ala Val Gly Ala Tyr Asp Val Ile                85                  90                  95 gtg aac ggggac aag gcc aac gtg ctc ttc cgg tgt gac aac ccc gac 336 Val Asn Gly AspLys Ala Asn Val Leu Phe Arg Cys Asp Asn Pro Asp            100                 105                 110 ggc aac tgt gctttg gaa ggc tgg ggc ggc cac tgg cgc ggc gcg aac 384 Gly Asn Cys Ala LeuGlu Gly Trp Gly Gly His Trp Arg Gly Ala Asn        115                 120                 125 gcc acc tcc gaa accgtc atc tgt gat cgc agc tac acc acc cgc cgc 432 Ala Thr Ser Glu Thr ValIle Cys Asp Arg Ser Tyr Thr Thr Arg Arg    130                 135                 140 tgg ctt gtc tcc atg tgctcc cag ggc tac acc gtc gcc ggc tcc gag 480 Trp Leu Val Ser Met Cys SerGln Gly Tyr Thr Val Ala Gly Ser Glu145                 150                 155                 160 acc aacacc ttc tgg gct tcg gac ctg atg cac cgt ctg tac cat gtg 528 Thr Asn ThrPhe Trp Ala Ser Asp Leu Met His Arg Leu Tyr His Val                165                 170                 175 cct gct gtgggt caa ggc cgg gtc gac cac ttc gcc gac ggc tac gac 576 Pro Ala Val GlyGln Gly Arg Val Asp His Phe Ala Asp Gly Tyr Asp            180                 185                 190 gag gtg att gccctg gcc aag agc aac ggc acc gag tcc acg cat gac 624 Glu Val Ile Ala LeuAla Lys Ser Asn Gly Thr Glu Ser Thr His Asp        195                 200                 205 tcg gag gcg ttg cagtat ttc gcc ctt gag gcg tat gcg ttt gat att 672 Ser Glu Ala Leu Gln TyrPhe Ala Leu Glu Ala Tyr Ala Phe Asp Ile    210                 215                 220 gcc gct ccc ggt gtc ggatgt gct ggc gag agt cac ggc cct gac cag 720 Ala Ala Pro Gly Val Gly CysAla Gly Glu Ser His Gly Pro Asp Gln225                 230                 235                 240 gga catgac acc ggg tct gcc tcg gcg cct gcg tct acc tcc acc tct 768 Gly His AspThr Gly Ser Ala Ser Ala Pro Ala Ser Thr Ser Thr Ser                245                 250                 255 agc tcc agctcg ggc tcg ggc tcg ggc gcc acg act acc ccg acg gat 816 Ser Ser Ser SerGly Ser Gly Ser Gly Ala Thr Thr Thr Pro Thr Asp            260                 265                 270 tct ccc agt gccact att gat gtg ccg tcg aac tgc cat acc cat gaa 864 Ser Pro Ser Ala ThrIle Asp Val Pro Ser Asn Cys His Thr His Glu        275                 280                 285 ggt gga cag ctt cattgc act 885 Gly Gly Gln Leu His Cys Thr     290                 295

A 16 amino acid signal sequence was predicted by PSORTII(http://psort.nibb.ac.jp/cgi-bin). The signal sequence was subsequentlyremoved as PSORTII predicted that the protein would be extracellular,which suggested that the protein would be secreted by the S. cerevisiaeif the signal sequence was left intact. A methionine was added beforethe 17th amino acid to ensure that the protein would be translated.

The following conditions were used to amplify AspF 2 from an Aspergillusfumigatus cDNA library: 25 μL reactions were performed in PCR Ready Beadtubes (Amersham Pharmacia) containing 500 ng of cDNA library, 2 μMprimers, and 13 μL sterile water. Reactions were performed using thePerkin Elmer 2400 Thermocycler (PE Applied Biosystems) under thefollowing conditions: an initial cycle at 94° C. for 5 min, 30 cycles at94° C. for 1 min, 50° C. for 1 min and 72° C. for 1 min, and a finalextension at 72° C. for 7 min.

The PCR reactions were separated using agarose gel electrophoresis and aphotograph of a representative gel data not shown. A band ofapproximately 936 bases corresponding to the amplified AspF 2 ORFcontains a 3′ c-myc tag.

The band was excised from the gel, purified and ligated into pCR 2.1.The ligated vector was used to transform competent E. coli cells andvector DNA isolated from 5 mL mini cultures using standard alkalinelysis techniques. DNA isolated from several miniprep isolates wasdigested with EcoRI and separated by agarose gel electrophoresis toconfirm the presence of the 936 base AspF 2/c-myc insert.

Several isolates were grown, cells harvested, plasmid DNA isolated andthe DNA sequence of the AspF 2 insert determined. A comparison of thesequence obtained empirically with the published sequence was determined(SEQ ID NOs: 5 and 6). There were no apparent PCR errors.

SEQ ID NO: 5 - AspF2 accaacacct tctgggcttc ggacctgatg caccgtctgtaccatgtgcc tgctgtgggt  60 caaggccggg tcgaccactt cgccgacggc tacgacgaggtgattgccct ggccaagagc 120 aacggcaccg agtccacgca tgactcggag gcgttgcagtatttcgccct tgaggcgtat 180 gcgtttgata ttgccgctcc cggtgtcgga tgtgctggcgagagtcacgg ccctgaccag 240 ggacatgaca ccgggtctgc ctcggcgcct gcgtctacctccacctctag ctccagctcg 300 ggctcgggct cgggcgccac gactaccccg acggattctcccagtgccac tattgatgtg 360 ccgtcgaact gccataccca tgaaggtgga cagcttcattgcactgaaca gaagttgatt 420 tccgaagaag acctcgag 438 SEQ ID NO: 6 - AspF2PCR accaacacct tctgggcttc ggacctgatg caccgtctgt accatgtgcc tgctgtgggt 60 caaggccggg tcgaccactt cgccgacggc tacgacgagg tgattgccct ggccaagagc120 aacggcaccg agtccacgca tgactcggag gcgttgcagt atttcgccct tgaggcgtat180 gcgtttgata ttgccgctcc cggtgtcgga tgtgctggcg agagtcacgg ccctgaccag240 ggacatgaca ccgggtctgc ctcggcgcct gcgtctacct ccacctctag ctccagctcg300 ggctcgggct cgggcgccac gactaccccg acggattctc ccagtgccac tattgatgtg360 ccgtcgaact gccataccca tgaaggtgga cagcttcatt gcactgaaca gaagttgatt420 tccgaagaag acctcgag 438

The DNA of an isolate was excised from the pCR 2.1 vector (Invitrogen)with a BamHI/EcoRI double digest and the reactions were separated byelectrophoresis so that the DNA band encoding the AspF 2 ORF could beisolated. This DNA fragment was then ligated into the pYEX-BX vectorusing the same restriction enzymes. The ligated vector was then used totransform competent E. coli and the plasmid DNA isolated by standardalkaline lysis mini-preps. The DNA sequence of salient portions of thevector was determined to ensure the correct alignment of the ORF withthe TATA box. Then portions of the vector were sequenced data not shown.The TATA box, the aspf-2 coding sequence, the c-myc tag sequence, andthe stop codons were identified (SEQ ID NOs:1 and 2).

Exemplary cloning method for Hemolysin Published DNA and predicted aminoacid sequences of Hemolysin to design PCR primers to amply the ORF fromthe A. fumigatus cDNA library were used. The DNA and amino acidsequences are presented in diagram form in SEQ ID NOs:7 and 8, alongwith the positions of the 5′ and 3′ primers used to obtain the predictedPCR product are shown. No signal sequence was predicted by PSORTII(http://psort.nibb.ac.jp/cgi-bin).

The following conditions were used to amplify hemolysin from the cDNAlibrary: 25 μL, reactions were performed in PCR Ready Bead tubescontaining 500 ng of cDNA library, 2 μM primers, and 13 μL, sterilewater. Reactions were performed using a Perkin Elmer 2400 Thermocyclerunder the following conditions: an initial cycle at 94° C. for 5 min, 30cycles at 94° C. for 1 min, 50° C. for 1 min, 72° C. for 1 min, and afinal extension at 72° C. for 7 min.

The PCR reactions were separated using agarose gel electrophoresis and aband of approximately 444 by corresponding to the amplified hemolysinORF was excised from the gel, purified and ligated into pCR 2.1. Theligated vector was used to transform competent E. coli cells and vectorDNA was subsequently isolated using standard techniques. DNA isolatedfrom several isolates was used as template for PCR reactions to confirmthe presence of the hemolysin insert. Not all isolates contained thehemolysin insert due to self ligation of the pCR 2.1 vector. See FIG. 5.

Several isolates were grown, cells harvested, plasmid DNA isolated andthe DNA sequence of the hemolysin were determined. A comparison thesequence obtained empirically with the published sequence revealed thatno PCR errors were apparent, the sequence as shown in SEQ ID NOs:9 and10.

The DNA of one isolate was treated with BamH I/EcoRI and the DNA bandencoding the hemolysin ORF/c-myc tag was isolated. This DNA fragment wasthen ligated into vector pYEX-BX that had also been digested with BamHI/EcoRI. The ligated vector was then used to transform competent E. coliand the plasmid DNA was subsequently isolated. The DNA sequence of therelevant portions of the vector was determined to ensure the correctalignment of the ORF with the TATA box and the c-myc tag was in framewith the rest of the ORF. The sequenced portion of the vector ispresented in SEQ ID NO:11. In addition, this vector was used forExamples 2 and 3.

In one exemplary method an empty vector control was generated aspreviously described in Methods (.This template was amplified by PCR asdiscussed previously. DNA isolated from several isolates was used astemplate for PCR reactions to confirm the presence of the c-myc insert.(SEQ ID NOs:1 and 2) The DNA of one isolate was treated with BamHI/EcoRI and the DNA band encoding the c-myc tag was isolated. This DNAfragment was then ligated into pYEX-BX which had also been digested withBamHI and EcoRI. The ligated vector was used to transform competent E.coli cells and vector DNA isolated using standard techniques. DNAisolated from several miniprep isolates was used as template for PCRreactions to confirm the presence of the c-myc control insert. (SEQ IDNOs:1 and 2)

Several isolates were grown, cells harvested, plasmid DNA isolated andthe DNA sequence of the c-myc control insert were determined. The DNAsequence of pertinent portions of the vector was determined to ensurethe correct alignment of the c-myc control with the TATA box.

In one exemplary method, transformants expressing each A. fumigatusprotein were identified and the cleared cell lysates were generated andstored as previously discussed.

After gel separation, expression of A. fumigatus proteins was determinedby western blot by methods known in the art. In one exemplary methodgrowth of transformants were determined. Transformants that weredetermined to produce a protein of the correct molecular weight abovewere grown in YNB minus uracil for 2-3 days at 30° C. with shaking Oncethe cultures were in stationary phase, the cells were counted and frozenin 50% glycerol at a final concentration of 1×108 c/mL (10 yeast units)and stored at −80° C. until use.

The production of vaccines for animal models was used as previouslydisclosed. Each of four A. fumigatus genes from a cDNA library usinggene-specific PCR primers were cloned. The DNA encoding each ORF wassequenced to confirm that the correct gene had been cloned followed byan inframe c-myc tag and that no PCR errors were introduced. The ORF ofeach gene was inserted into the copper inducible vector pYEX-BX.Portions of each ligated vector were sequenced to ensure the correctposition of the gene relative to the TATA box.

Each pYEX-BX vector containing a gene of interest was used to transformyeast and yeast transformants expressing each of the four proteinsidentified by Western blot analysis. Each S. cerevisiae strain thatproduced the correct protein was subsequently produced and used tovaccinate an animal as discussed above.

Aspergillus. In another exemplary method, the protective efficacy ofHEMO antigen expressed in Saccharomyces spp. against systemicaspergillosis was tested in mice.

Antigen. HEMO antigen was administered s.c. using two injection sitesdorsally (0.075 ml each) on days 28, 21 and 14 before infection,receiving 6×10⁷ yeast cells per animal. Control animals received PBS(diluent control) or MYC (yeast not expressing immunogen control) at6×10⁷ yeast cells per animal.

Animals. Thirty six weeks-old male CD-1 mice were used. Animals werehoused in standard conditions in cages of 5 animals. Groups of 10 miceper group were established.

Infection. A total of 5.8×106 viable Aspergillus fumigatus conidia permouse were inoculated intravenously via lateral tail vein.

Tissue burden. 16 days after infection surviving animals wereeuthanatized by CO2 anoxia. The kidney and brain were removedaseptically, homogenized in 0.9% saline, diluted and plated on SDAplates for CFU determination.

Statistics. Comparisons of survival were done by a log rank test andtissue burden by Mann Whitney test using GraphPad 3.03 for Windows.

FIG. 12 represents an exemplary Log 10 CFU per studied organ.Pretreatment with HEMO antigen significantly reduced tissue burdens frombrain and kidney in comparison to the diluent control (p=0.003 and0.023, respectively). In addition the yeast control (MYC) reduced organsburdens as well but did not reach statistical significance (p=0.089 inboth organs). No differences were found between MYC and PBS groups.Importantly, 50 and 60% of the animals receiving MYC or HEMO,respectively, did not show detectable amount of A. fumigatus in brainbut all PBS-pretreated animals showed fungal grown in this organ.

The protective effect of the A. fumigatus antigen (HEMO) expressed inSaccharomyces was tested in a systemic murine model of aspergillosis.The MYC only yeast control group did show a reduction in the brain organburden. Importantly, the tissue burden study demonstrated that animalsreceiving three doses of the HEMO antigen before the infection hadstatistically significantly lower CFU of the fungus in the kidneys andbrain in comparison with the PBS-treated control animals. These resultsshow that the vaccine containing either yeast alone (MYC) or Hemoprotected mice against an infection of Aspergillus fumigatus.

Example 2

In certain exemplary methods, the fungal organism Coccidioides wasexamined.

See one exemplary schematic for production of and testing for ananti-fungal vaccination in a Coccidioides animal model (FIG. 6). In oneexemplary method, the feasibility of using heat-killed yeast cellsexpressing a C. immitis antigen as a vaccine against C. immitisinfections was examined. cDNA was obtained for each the C. immitis genewhose protein has been shown in other work to elicit an immune responsein infected animals. Then yeast were transformed with each cDNA andstrains selected that expressed a high levels of the antigen. Aschematic of the strategy is depicted in FIG. 6.

In another exemplary method, the protective effect of Coccidioidesimmitis antigen expressed by Saccharomyces cerevisiae was analyzed andused as a killed whole vaccine preparation in a systemic murine model ofcoccidioidomycosis. DNA encoding for Ag2 (also called PRA) was clonedinto S. cerevisiae and gene expression induced using for example,copper. These yeast cells were heat killed and used to vaccinate miceprior to infection with Coccidioides immitis.

Animals. Six-week-old male CD-1 mice were purchased from Charles RiverLaboratories. Groups consisted on 20 mice, housed in standardconditions. After infection, mice were housed in micro-isolator cages.Animals were provided food and acidified water ad libitum.

Antigen. In this exemplary example, a C. immitis antigen expressed inSaccharomyces was used. This was a protein present in the alkali-solubleand water-soluble preparation of the spherule wall and called antigen 2(Ag2/PRA). The vaccine was administered subcutaneously (s.c.) in avolume of 50 μl three times before infection (i.e., 21, 14 and 7 daysprior to the infection) at a concentration of 4×10⁸ cells/ml. In thisexample, PBS was used as the control.

Inoculum. Coccidioides immitis (Silveira strain) was used. The strainwas grown on glucose yeast-extract (GYE) plates in a Class 3 biosafetycabinet at room temperature for 2 weeks. Arthroconidia were harvested asa suspension in saline. The number of arthroconidia was determined byhemacytometer and verified for viability by cfu (colony forming units)determination done by plating inoculum dilutions on GYE medium. Animalswere infected intravenously (i.v.) with a suspension containing 376viable arthroconidia per mouse. Statistical analysis of survival wasdone using a log rank test.

FIG. 7 illustrates an exemplary the survival curve of the groups of miceincluded in the study that was vaccinated with yeast expressing theantigen-2/PRA protein. Deaths of control animals and pretreated animalsbegan on day 13 after infection and by day 21 only 10% of the PBStreated mice were still alive. This was in contrast to the mice treatedwith the Ag-2 containing yeast (25% still alive) Statistical comparisonsbetween groups showed a significant difference between the control groupand Ag2 immunized animals (p=0.04). This example shows that micevaccinated with yeast cells expressing the antigen-2/PRA proteinsurvived to a greater extent than untreated mice.

FIG. 8 illustrates an exemplary a Western Blot of extracts of yeastscontaining and expressing Hemolysin (hemo), aspf 2, or antigen 2 (theCoccidioides protein described above). The numbers indicate the amountof lysate separated by SDS-GEL electrophoresis. The blots were treatedwith a c-myc epitope specific antibody and the antibody detected using asecondary horse radish peroxidase antibody. Various amounts inmicrograms (indicated by the numbers at the bottom of the figure) ofc-myc peptide were spotted.

Antigen 2/proline-rich antigen has been previously shown to beprotectivein murine models of coccidiomycosis. Thus antigen 2 waschosen. Published DNA and predicted amino acid sequences were used todesign PCR primers to amplify the ORF from the Cox cDNA library (SEQ IDNOs:12 and 13). The DNA and amino acid sequences of antigen 2 arepresented in diagram form, data not shown. An 18 amino acid signalsequence was predicted by PSORTII (http://psort.nibb.ac.jp/cgi-bin), butwas not removed as PSORTII also predicted that the protein would be cellwall bound, most likely GPI anchored. This prediction suggests that theprotein would not be secreted by the S. cerevisiae.

Exemplary conditions used to amplify antigen 2 from the cDNA librarywere as follows: 25 μL, reactions were performed in PCR Ready Bead tubes(Amersham Pharmacia) containing 500 ng of cDNA library, 2 μM primers,and 13 μL sterile water. Reactions were performed using the Perkin Elmer2400 Thermocycler (PE Applied Biosystems) under the followingconditions: an initial cycle at 94° C. for 5 min, 30 cycles at 94° C.for 1 min, 50° C. for 1 min and 72° C. for 1 min, and a final extensionat 72° C. for 7 min. As previously discussed, PCR reactions wereseparated and a band of approximately 582 bases corresponding to theamplified antigen 2 ORF was excised. The band was excised from the gel,purified and ligated into pCR 2.1. The ligated vector was used totransform competent E. coli cells and vector DNA isolated from 5 mL minicultures using standard alkaline lysis techniques. DNA isolated fromseveral miniprep isolates was digested with EcoRI to confirm thepresence of the 582 base antigen 2 insert. Only one isolate containedthe antigen 2 insert.

Several isolates were grown, cells harvested, plasmid DNA isolated andthe DNA sequence of the antigen 2 insert determined. A comparison of thesequence obtained empirically with the published sequence is shown inSEQ ID NOs:14 and 15. There were no apparent PCR errors.

SEQ ID NO: 14: Antigen-2ATGCAGTTCTCTCACGCTCTCATCGCTCTCGTCGCTGCCGGCCTCGCCAGTGCCCAGCTCCCAGACATCCCACCTTGCGCTCTCAACTGCTTCGTTGAGGCTCTCGGCAACGATGGCTGCACTCGCTTGACCGACTTCAAGTGCCACTGCTCCAAGCCTGAGCTCCCAGGACAGATCACTCCTTGCGTTGAGGAGGCCTGCCCTCTCGACGCCCGTATCTCCGTCTCCAACATCGTCGTTGACCAGTGCTCCAAGGCCGGTGTCCCAATTGACATCCCACCAGTTGACACCACCGCCGCTCCCGAGCCATCCGAGACCGCTGAGCCCACCGCTGAGCCAACCGAGGAGCCCACTGCCGAGCCTACCGCTGAGCCCACCGCTGAGCCGACTCATGAGCCCACCGAGGAGCCCACTGCCGTCCCAACCGGCACTGGCGGTGGTGTCCCCACTGGCACCGGTTCCTTCACCGTCACTGGCAGACCAACTGCCTCCACCCCAGCTGAGTTCCCAGGTGCTGGCTCCAACGTCCGTGCCAGCGTTGGCGGCATTGCTGCTGCTCTCCTCGGTCTCGCTGCCTACCTG SEQ ID NO: 15: Antigen-2 PCRATGCAGTTCTCTCACGCTCTCATCGCTCTCGTCGCTGCCGGCCTCGCCAGTGCCCAGCTCCCAGACATCCCACCTTGCGCTCTCAACTGCTTCGTTGAGGCTCTCGGCAACGATGGCTGCACTCGCTTGACCGACTTCAAGTGCCACTGCTCCAAGCCTGAGCTCCCAGGACAGATCACTCCTTGCGTTGAGGAGGCCTGCCCTCTCGACGCCCGTATCTCCGTCTCCAACATCGTCGTTGACCAGTGCTCCAAGGCCGGTGTCCCAATTGACATCCCACCAGTTGACACCACCGCCGCTCCCGAGCCATCCGAGACCGCTGAGCCCACCGCTGAGCCAACCGAGGAGCCCACTGCCGAGCCTACCGCTGAGCCCACCGCTGAGCCGACTCATGAGCCCACCGAGGAGCCCACTGCCGTCCCAACCGGCACTGGCGGTGGTGTCCCCACTGGCACCGGTTCCTTCACCGTCACTGGCAGACCAACTGCCTCCACCCCAGCTGAGTTCCCAGGTGCTGGCTCCAACGTCCGTGCCAGCGTTGGCGGCATTGCTGCTGCTCTCCTCGGTCTCGCTGCCTACCTG

The DNA of the isolate was excised from the pCR 2.1 vector and thesalient portions of the vector was determined to ensure the correctalignment of the ORF with the TATA box and the c-myc tag, as describedpreviously. Published DNA and predicted amino acid sequences were usedto design PCR primers to amply the ORF from the cDNA library for antigen2.

In one exemplary method an empty vector control for animal models wasdesigned as previously indicated; that is, the pYEX-BX vector containingthe c-myc tag but lacking a C. immitis gene of interest. Severalisolates were grown, cells harvested, plasmid DNA isolated and the DNAsequence of the c-myc control insert were determined. The DNA sequenceof pertinent portions of the vector was determined to ensure the correctalignment of the c-myc with the TATA box.

Transformation of S. cerevisiae was performed as previously described.After obtaining at least 10 transformants for each C. immitis gene ofinterest, cells are grown and induced the expression of each gene byadding copper sulfate. Uninduced and induced cells were harvested bycentrifugation, lysed in SDS-gel lysis buffer and the proteinconcentration of each extract determined. Equal protein amounts of eachlysate were separated by SDS-PAGE and the separated proteins transferredto membranes. The presence of the c-myc tagged proteins was detected byWestern blot analysis using an anti-c-myc antibody. Then thetransformants were grown as previously described. Once transformants ofeach construct were isolated, the proteins were expressed via copperinduction as previously discussed. Then cultures were grown overnight at30° C. with shaking. These cultures were used for the identification oftransformants that were expressing each of the C. immitis proteins.

Identification of transformants expressing each C. immitis protein.Induced and uninduced cultures were harvested after overnight incubationby centrifugation at 550×g for 10 minutes. Each cell pellet was washedwith 25 mL ice cold water to remove any residual medium and the cellswere harvested again by centrifugation. Each pellet was resuspended in 1mL of ice cold water and divided into two 1.5 mL eppendorf tubes forcentrifugation. After the removal of the supernatant, one tube wasfrozen at −80° C. for storage and the other was lysed for proteindetermination. Cells were lysed in the following manner: 100 μL of glassbeads were added to the tube containing cells followed by the additionof 200 μL 2× sample lysis buffer (0.1M Tris, pH 6.8, 20% (v/v) glycerol,4% (w/v) SDS, 6.2 mg DTT). Tubes were mixed by vortexing for 1 min andboiled for 3 min to resuspend and lyse the cells. Debris was removed bycentrifugation and the cleared supernatant was transferred to a newtube. The cleared lysate was used for western blots.

Cell lysates were loaded into the wells of SDS page gels and expressionof the C. immits proteins were determined by exemplary western blotprocedures known in the art. Each of three C. immitis genes from a cDNAlibrary using gene-specific PCR primers was obtained. The DNA encodingeach ORF was sequenced to confirm that the correct gene had been clonedand that no PCR errors were introduced. The ORF of each gene wasinserted into the copper inducible vector pYEX-BX. Each ligated vectorwas sequenced to ensure the correct position of the gene relative to theTATA box and that the c-myc tag was in frame with each protein.

Example 3

In certain exemplary methods, the fungal organism Cryptococcosis wasanalyzed (see exemplary schematic FIG. 9)

Cryptococcosis model. In one exemplary method, an inhalation model ofcryptococcosis was chosen because 1) it most closely mimics the naturalcourse of C. neoformans infection, 2) it is highly reproducible and 3)it is commonly used to assess virulence. The inhalation cryptococcosismodel performed was essentially as previously described (see: Cox, G.M., Mukherjee, J., Cole, G. T., et al. Urease as a virulence factor inexperimental cryptococcosis. Infect Immun. (2000) 68:443-448.incorporated herein by reference).

To test the in vivo efficacy of each vaccine formulation to protectvaccinated animals against a challenge of C. neoformans, mice arevaccinated with various vaccine formulations, and challenged with C.neoformans, and monitored for protection.

Organism. C. neoformans H 99 was used as the challenge organism for allinfections. C. neoformans H99 yeast for the inoculum was prepared asfollows. Briefly, C. neoformans strain H99 was grown at 30° C. withshaking for two days in liquid YPD medium. The cells were harvested bycentrifugation, washed in endotoxin-free PBS, and resuspended inendotoxin-free PBS. The cells were counted on a hemocytometer anddiluted to 1×10⁶ cells per ml. The inoculum was serially diluted andplated onto YPD to confirm the number of cells that are inoculated.

Mice. Four-week-old female A/J Cr mice were obtained from Charles RiverLaboratories for the inhalation model of cryptococcosis. Mice arecommonly used for virulence assays with the serotype A strain, H99, andhave been used in previous assessments of antibody protection against C.neoformans.

Briefly, mice from a specific pathogen-free colony were vaccinated with150 μL of each yeast vaccine preparation on day 21, 14 and 7 beforebeing challenged with 50 uL containing 5×10⁴H99 C. neoformans cells.

In one exemplary method, experiments were performed to determine whetheryeast strains expressing C. neoformans proteins could be used as avaccine against C. neoformans infections. cDNA of certain genes whoseproteins are likely to be important in infection were generated. Thenyeast cells were transformed with each cDNA and select strains thatexpress high levels of each putative antigen. Finally, each vaccineformulation was tested for its ability to protect vaccinated miceagainst a challenge of C. neoformans. FIG. 9 represents a schematic ofthese experiments.

DNA vector construction and Yeast Transformation were performed in thisexample as previously described. Primers are listed in Table 4 (SEQ IDNOs: 30-41). Then the cloned genes were used as template and amplifiedeach gene using the gene-specific PCR primers. The DNA sequence of eachligated pYEX-BX vector containing a gene of interest was determined toensure that the correct gene was cloned, the start codon was on the sameDNA strand as the TATA box (initiation of transcription), and that thec-myc tag was in frame with each gene followed by two stop codons (TGATAA).

Cloning of CDA: Published DNA was used and predicted amino acidsequences to design PCR primers to amplify the ORF from the clone, asdescribed above. The DNA and amino acid sequences of CDA are presentedin diagram form in SEQ ID NOs: 16-17. The positions of the 5′ and 3′primers use to obtain the predicted PCR product are shown. A 19 aminoacid signal sequence was predicted by PSORTII(http://psort.nibb.ac.jp/cgi-bin). The signal sequence was subsequentlyremoved as PSORTII predicted that the protein would be extra-cellular,which suggested that the protein would be secreted by the S. cerevisiaeif the signal sequence was left intact. A methionine was added beforethe 20th amino acid (Serine) to ensure that the protein would betranslated.

SEQ ID NOs: 16 and 17 - DNA and corresponding amino acid sequence of CDAatg aag ttc atc aca agc ctc ttt gcc gtt ctt gcc att ctc tca agt  48 MetLys Phe Ile Thr Ser Leu Phe Ala Val Leu Ala Ile Leu Ser Ser1               5                   10                  15 gtc tct gcttct cct acc atg aag aaa cgt gcg acc gtc gaa act atc  96 Val Ser Ala SerPro Thr Met Lys Lys Arg Ala Thr Val Glu Thr Ile            20                  25                  30 aac aac tgt aatcag cag ggc act gtt gct ctg acc ttt gac gat ggc 144 Asn Asn Cys Asn GlnGln Gly Thr Val Ala Leu Thr Phe Asp Asp Gly        35                  40                  45 cct tac aat tac gaagcc caa gtt gct tct gcc ctt gac ggg ggt aag 192 Pro Tyr Asn Tyr Glu AlaGln Val Ala Ser Ala Leu Asp Gly Gly Lys    50                  55                  60 ggt act ttt ttc ctc aacggc gcg aat tat gtc tgc atc tac gac aag 240 Gly Thr Phe Phe Leu Asn GlyAla Asn Tyr Val Cys Ile Tyr Asp Lys65                  70                  75                  80 gcc gatgaa atc aga gct ttg tat gat gcc ggc cac act ctt ggt tct 288 Ala Asp GluIle Arg Ala Leu Tyr Asp Ala Gly His Thr Leu Gly Ser                85                  90                  95 cac act tggtct cac gcc gac ctt acc cag tta gat gaa tcc ggg atc 336 His Thr Trp SerHis Ala Asp Leu Thr Gln Leu Asp Glu Ser Gly Ile            100                 105                 110 aac gag gaa ctctcc aag gtc gaa gat gcc ttt gtc aag atc ctt ggt 384 Asn Glu Glu Leu SerLys Val Glu Asp Ala Phe Val Lys Ile Leu Gly        115                 120                 125 gtc aag cct cga tacttc cga ccc cct tac ggt aac atc aac gac aac 432 Val Lys Pro Arg Tyr PheArg Pro Pro Tyr Gly Asn Ile Asn Asp Asn    130                 135                 140 gtc ttg aac gtc ctc agtgaa agg ggt tac acg aag gtg ttt ttg tgg 480 Val Leu Asn Val Leu Ser GluArg Gly Tyr Thr Lys Val Phe Leu Trp145                 150                 155                 160 tct gatgac act ggg gat gcc aac ggc gag tcg gtc agt tac tcc gag 528 Ser Asp AspThr Gly Asp Ala Asn Gly Glu Ser Val Ser Tyr Ser Glu                165                 170                 175 ggg gta ttggac aac gtt atc cag gat tat cct aac cct cat ctt gtc 576 Gly Val Leu AspAsn Val Ile Gln Asp Tyr Pro Asn Pro His Leu Val            180                 185                 190 ctt gat cac tctacc atc gag acg acc tcc tcc gag gtt ctc cct tac 624 Leu Asp His Ser ThrIle Glu Thr Thr Ser Ser Glu Val Leu Pro Tyr        195                 200                 205 gct gta ccc aag ctccag agt gct ggc tac caa ctg gtc act gtc ggt 672 Ala Val Pro Lys Leu GlnSer Ala Gly Tyr Gln Leu Val Thr Val Gly    210                 215                 220 gaa tgt ctc ggc acc gacgaa tct cct tac gaa tgg gtt gat tgc cct 720 Glu Cys Leu Gly Thr Asp GluSer Pro Tyr Glu Trp Val Asp Cys Pro225                 230                 235                 240 gga gagagg gat agc tct tgg caa tgc 747 Gly Glu Arg Asp Ser Ser Trp Gln Cys                245

The following conditions were used to amplify CDA from the plasmid: 25μL reactions were performed in PCR Ready Bead tubes (Amersham Pharmacia)containing 500 ng of the plasmid containing CDA, 2 μM primers, and 13 μLsterile water. Reactions were performed using the Perkin Elmer 2400Thermocycler (PE Applied Biosystems) under the following conditions: aninitial cycle at 94° C. for 5 min, 30 cycles at 94° C. for 1 min, 50° C.for 1 min and 72° C. for 1 min, and a final extension at 72° C. for 7min.

The PCR reactions were separated using agarose gel electrophoresis and aband of approximately 744 bases corresponding to the amplified CDA ORFcontaining a 3′ c-myc tag was excised. The band was purified and ligatedinto pCR 2.1. The ligated vector was used to transform competent E. colicells and vector DNA isolated from 5 mL mini cultures using standardalkaline lysis techniques. DNA isolated from several miniprep isolateswas digested with EcoRI to confirm the presence of the 744 baseCDA/c-myc insert.

In one exemplary method, the DNA of the isolate was excised from the pCR2.1 vector and ligated into the pYEX-BX vector as previously describedand the salient portions of the vector was determined to ensure thecorrect alignment of the ORF with the TATA box

One exemplary method concerns cloning of Laccase: As indicated above,the same strategy to clone Laccase (lac) from a plasmid containing thegene as used to clone CDA. DNA sequence and plasmid DNA was used topredicted the amino acid sequences to design PCR primers to amplify theORF plus the c-myc tag from the plasmid. The DNA and amino acidsequences are presented in diagram form in SEQ ID NOs:18 and 19. Thepositions of the 5′ and 3′ primers used to obtain the predicted PCRproduct are shown. A 20 amino acid signal sequence was predicted byPSORTII (http://psort.nibb.ac.jp/cgi-bin). The signal sequence wassubsequently removed as PSORTII predicted that the protein would beextracellular, which suggested that the protein would be secreted by theS. cerevisiae if the signal sequence was left intact. An endogenousmethionine at amino acid 21 (Glutamic acid) was used as the first aminoacid in the new protein to ensure that the protein would be translated.

atg cgg gga gta gtc aag ctc ttc ttt cta tct tgt tcc ctc gtt tcg   48 MetArg Gly Val Val Lys Leu Phe Phe Leu Ser Cys Ser Leu Val Ser1               5                   10                  15 ctg gtc agcagc gag gag act ggc aag tcg cca acc gcg aac tat gac   96 Leu Val Ser SerGlu Glu Thr Gly Lys Ser Pro Thr Ala Asn Tyr Asp            20                  25                  30 cat tat atg ccgaag gcg aca gca acc att gat cct agt gta ttc gct  144 His Tyr Met Pro LysAla Thr Ala Thr Ile Asp Pro Ser Val Phe Ala        35                  40                  45 ctt tca aat gac tttgaa ata aca gat gtt ccg acg acg agg gag tat  192 Leu Ser Asn Asp Phe GluIle Thr Asp Val Pro Thr Thr Arg Glu Tyr    50                  55                  60 acc ttc gat atc acc aaagcg ttg gcc agc cct gat ggt tat gaa cga  240 Thr Phe Asp Ile Thr Lys AlaLeu Ala Ser Pro Asp Gly Tyr Glu Arg65                  70                  75                  80 gag gtttac gtt gtc aac aac atg ttc cct gga cct gtg ata gag gct  288 Glu Val TyrVal Val Asn Asn Met Phe Pro Gly Pro Val Ile Glu Ala                85                  90                  95 aac acc ggggat act att atc gta cat gtc aac aat cat ttg gag gaa  336 Asn Thr Gly AspThr Ile Ile Val His Val Asn Asn His Leu Glu Glu            100                 105                 110 gga caa agt atccac tgg cat ggt ttg cgg cag ctt ggc acg gct ttc  384 Gly Gln Ser Ile HisTrp His Gly Leu Arg Gln Leu Gly Thr Ala Phe        115                 120                 125 atg gac ggt gtc cctggt ata aca cag tgt cct att ccc cct gga agc  432 Met Asp Gly Val Pro GlyIle Thr Gln Cys Pro Ile Pro Pro Gly Ser    130                 135                 140 tca ttt acc tac caa ttcacc gta agc cat cag tca ggc acg ttt tgg  480 Ser Phe Thr Tyr Gln Phe ThrVal Ser His Gln Ser Gly Thr Phe Trp145                 150                 155                 160 tgg cattcc cat tat tcc aat tcc atg gcc gac ggc att tgg ggc ccc  528 Trp His SerHis Tyr Ser Asn Ser Met Ala Asp Gly Ile Trp Gly Pro                165                 170                 175 tta att atccat tcg ccc aat gaa ccc ctc caa agg gga cga gac tat  576 Leu Ile Ile HisSer Pro Asn Glu Pro Leu Gln Arg Gly Arg Asp Tyr            180                 185                 190 gac gag gat cgaatc gtt ttt ata act gac tgg gtg cat gac aac tca  624 Asp Glu Asp Arg IleVal Phe Ile Thr Asp Trp Val His Asp Asn Ser        195                 200                 205 gaa gtc gtt att gcagct cta gct act cca gaa ggg tac aaa gga agc  672 Glu Val Val Ile Ala AlaLeu Ala Thr Pro Glu Gly Tyr Lys Gly Ser    210                 215                 220 cct gct ccg cca caa ggtgat gcg att ctc atc aat gga cgt ggc caa  720 Pro Ala Pro Pro Gln Gly AspAla Ile Leu Ile Asn Gly Arg Gly Gln225                 230                 235                 240 acc aactgc aca gcc act ggt tcc tcc tca tgc acc tat ccg cct cct  768 Thr Asn CysThr Ala Thr Gly Ser Ser Ser Cys Thr Tyr Pro Pro Pro                245                 250                 255 ccc gag attcac gtg cca gtc aat tgc agg gtt cgt ctg cgc ttt atc  816 Pro Glu Ile HisVal Pro Val Asn Cys Arg Val Arg Leu Arg Phe Ile            260                 265                 270 agt gcg acc gcccat ccc atg tac cgc ata act atc gac aac cac cct  864 Ser Ala Thr Ala HisPro Met Tyr Arg Ile Thr Ile Asp Asn His Pro        275                 280                 285 ttg gaa gtt gtg gaaacc gac ggt aca gcc gtc tat ggg ccc aca gtc  912 Leu Glu Val Val Glu ThrAsp Gly Thr Ala Val Tyr Gly Pro Thr Val    290                 295                 300 cat gaa atc tcc att gcacct ggg gaa cgg tac tct gca att atc aac  960 His Glu Ile Ser Ile Ala ProGly Glu Arg Tyr Ser Ala Ile Ile Asn305                 310                 315                 320 acc tcagaa ggg aag gaa ggt gat gcg ttc tgg ctg agg aca agt gtt 1008 Thr Ser GluGly Lys Glu Gly Asp Ala Phe Trp Leu Arg Thr Ser Val                325                 330                 335 gct ctg ggctgt atg ttt ggt gga ata gat cag gtg gga ttg gcg gtt 1056 Ala Leu Gly CysMet Phe Gly Gly Ile Asp Gln Val Gly Leu Ala Val            340                 345                 350 gtg agg tat acgggt aat gga atg gtt agt act gaa gag cct caa act 1104 Val Arg Tyr Thr GlyAsn Gly Met Val Ser Thr Glu Glu Pro Gln Thr        355                 360                 365 act gct tgg agt gatcta gcg gga gct aca act cct tgt gct gga ctg 1152 Thr Ala Trp Ser Asp LeuAla Gly Ala Thr Thr Pro Cys Ala Gly Leu    370                 375                 380 gac caa aca tat act ctttca cca cga gag agt ttt agt gca cct cgt 1200 Asp Gln Thr Tyr Thr Leu SerPro Arg Glu Ser Phe Ser Ala Pro Arg385                 390                 395                 400 gaa ttttca caa agc cat gtc ttc aat agc cag cga gga gcc ttt gtg 1248 Glu Phe SerGln Ser His Val Phe Asn Ser Gln Arg Gly Ala Phe Val                405                 410                 415 aat gtt tatggc aac acc ttc caa ggt tat ggg ttt aac aat atc tca 1296 Asn Val Tyr GlyAsn Thr Phe Gln Gly Tyr Gly Phe Asn Asn Ile Ser            420                 425                 430 tat cag aac caaatc ttc aac cct cta ctt tca atc gtc caa cgc ggt 1344 Tyr Gln Asn Gln IlePhe Asn Pro Leu Leu Ser Ile Val Gln Arg Gly        435                 440                 445 ggc tct tgc gag agcaca cta gta gcc agt aca act ttc ccc gac ctc 1392 Gly Ser Cys Glu Ser ThrLeu Val Ala Ser Thr Thr Phe Pro Asp Leu    450                 455                 460 gga tca ggg aac att atcatc aac aat ctt gat ggc gtt atc gac cat 1440 Gly Ser Gly Asn Ile Ile IleAsn Asn Leu Asp Gly Val Ile Asp His465                 470                 475                 480 cct taccac ctg cac ggc aac gag ttc cag gtg ata gga cga gga act 1488 Pro Tyr HisLeu His Gly Asn Glu Phe Gln Val Ile Gly Arg Gly Thr                485                 490                 495 gga gct ctcagc ctt gat aac ctg aca aat att gac ttc aat ttg gac 1536 Gly Ala Leu SerLeu Asp Asn Leu Thr Asn Ile Asp Phe Asn Leu Asp            500                 505                 510 aac cct gtg agaaag gat acc ctc tgg ata cag ggc gga agt tgg gtg 1584 Asn Pro Val Arg LysAsp Thr Leu Trp Ile Gln Gly Gly Ser Trp Val        515                 520                 525 gta ctg agg atc acgacg gat aac cct gga gtt tgg gcc ttg cac tgt 1632 Val Leu Arg Ile Thr ThrAsp Asn Pro Gly Val Trp Ala Leu His Cys    530                 535                 540 cat att ggg tgg cat cttact gag gga aag ttg gct gtg gtt gtc att 1680 His Ile Gly Trp His Leu ThrGlu Gly Lys Leu Ala Val Val Val Ile545                 550                 555                 560 caa ccaggt gcg att gga cat atg gag ggc ccc gag tct tgg acg aat 1728 Gln Pro GlyAla Ile Gly His Met Glu Gly Pro Glu Ser Trp Thr Asn                565                 570                 575 ctc tgt gctaac act gat ccc aat gca ttt gga ccc gca cga cgc tca 1776 Leu Cys Ala AsnThr Asp Pro Asn Ala Phe Gly Pro Ala Arg Arg Ser            580                 585                 590 cct tct cca tctatt caa tcc tct aag aca tcc act ttc cag tat ctg 1824 Pro Ser Pro Ser IleGln Ser Ser Lys Thr Ser Thr Phe Gln Tyr Leu        595                 600                 605 cgc gaa gtg aaa gggaag gtc gtt aaa cgt aga ggt gct cga gag gcg 1872 Arg Glu Val Lys Gly LysVal Val Lys Arg Arg Gly Ala Arg Glu Ala    610                 615                 620

The following conditions were used to amplify laccase from the plasmid:25 μL reactions were performed in PCR Ready Bead tubes containing 500 ngof plasmid containing Laccase, 2 μM primers, and 13 μL sterile water.Reactions were performed using a Perkin Elmer 2400 Thermocycler underthe following conditions: an initial cycle at 94° C. for 5 min, 30cycles at 94° C. for 1 min, 50° C. for 1 min, 72° C. for 1 min, and afinal extension at 72° C. for 7 min.

The PCR reactions were separated, isolated and digested with EcoRI aspreviously described to confirm the presence of the 1857 base Lac/c-mycinsert.

Several isolates were grown, cells harvested, plasmid DNA isolated andthe DNA sequence of the Lac insert determined. The DNA of the isolatewas excised from the pCR 2.1 vector and the DNA band encoding theLaccase ORF could be isolated, data not shown. This DNA fragment wasthen ligated into the pYEX-BX vector as described and then used totransform competent E. coli and the plasmid DNA isolated by standardalkaline lysis mini-preps. The DNA sequence of salient portions of thevector was determined to ensure the correct alignment of the ORF withthe TATA box.

An empty vector control was made and used as previously described inmethods.

Transformation of S. cerevisiae was made and used as previouslydescribed in methods.

Identification of yeast transformants expression each C. neoformansprotein. After obtaining transformed S. cerevisiae cells that containedeach gene of interest 5 transformants were screened for each gene ofinterest were screened to determine the expression of each gene and thetransformants were isolated as previously described. Once transformantscontaining each construct were isolated, each of the proteins wasexpressed as described previously. Then transformants expressing each C.neoformans protein were identified by one exemplary method previouslydisclosed.

Expression of C. neoformans proteins were determined by an exemplarywestern blot technique known in the art. Ten CDA transformants werescreened to obtain a transformant that was positive for induction viacopper. Transformant 10 was chosen for vaccine production as it had themost protein as determined by counting pixels. Five Lac transformantswere screened to obtain a transformant that was positive for inductionvia copper. Please note that the top band is of the correct size.Transformant number 2 had the least amount of protein in the un-inducedsample and the most (by counting pixels) in the copper induced sample

Growth of transformants: Transformants that were determined to produce aprotein of the correct molecular weight were grown in YNB minus uracilfor 2-3 days at 30° C. with shaking Once the cultures were in stationaryphase, the cells were counted and frozen in 50% glycerol at a finalconcentration of 1×108 c/mL (10 yeast units) and stored at −80° C. untiluse.

Yeast cells containing and expressing chitin deacetylase or laccase wereharvested and lysed as described. The indicated number of microliters ofeach extract were separated by SDS-PAGE, transfer to membranes and theposition of each protein detected by Western blot analysis as described.The results of the western blot are shown in FIG. 10. Lane 1-4 arevarious amounts in microliters of lysed yeast cells expressing chitindeacetylase (cda; MW. 26.6 kDa), while lanes 6-9 are various amounts inmicroliters of lysed yeast cells expressing laccase (lac; MW. 67.4 kDa).

FIG. 11 represents exemplary linear survival plots in an animal modelafter exposure to a challenge fungal organism. Mice were vaccinated withyeast cells containing and expressing the DNA encoding laccase, (lac),chitin deacetylase (cda), the MYC yeast control, PBS, trr and plb (twoadditional C. neoformans proteins) as described above on days 21, 14 and7 prior to challenge by C. neoformans H99 cells. Mice were weighed dailyand mice that had lost >15% of their body weight euthanized. Note thatmice vaccinated with lac-containing yeast were 100% protected (p<<0.05)while those vaccinated with cda containing yeast were 80% protected(p<0.05) as were those vaccinated with MYC (empty vector control). Theseresults demonstrate that mice vaccinated with yeast expressing eitherlaccase or chitin deacetylase were highly protected against a challengeof C. neoformans.

TABLE 1 Candidate antigens Number of Base pairs Protein amino acids ofthe ORF Accession numbers fos-1 708 2,124 Protein: AAK27436 DNA:AF257496 hemolysin 131 721 Protein: BAA03951.1 DNA: D16501 catalase 7282,184 Protein: AAB71223 DNA: U97574 abr-1 664 1,992 Protein: AAF03353DNA: AF116901 rod A 159 477 Protein: AAB60172.1 DNA: U06121 dpp-5 7212,163 Protein: AAB67282 DNA: L48074 dpp-4 765 2,295 Protein: AAC34310DNA: U87950

TABLE 2 Cryptococcus antigens Number of Base pairs Protein amino acidsof the ORF Accession numbers CDA - Chitan 250 750 Protein: CAD10036deacetylase DNA: AJ414580 CnFOS - 1367 4101 Protein: AAW42353 histidinekinase DNA: NA* Lac - Laccase 625 1875 Protein: NA* DNA: L22866 Plb -638 1914 Protein: AAF65220.1 phospholipse B DNA: AF223383 Ure - Urease833 2499 Protein: AAC62257.1 DNA: AF006062 *NA—not available

TABLE 3 PCR primers 5′ AspF2/Bam 5′GGA TCC ATG CTT GTG GCC ACC CTC CCT3′ (SEQ ID NO: 20) 3′ AspF2/myc 5′GAA TTC TTA TCA CTC GAG GTC TTC TTCGGA AAT CAA CTT AGT GCA ATG AAG CTG TCC ACC TTC ATG GGT ATG GC 3′ (SEQID NO: 21) 5′DppV/Bam 5′GGA TTC ATG CTT ACA CCT GAG CAG CTA ATC ACT GCTCCA CGG 3′ (SEQ ID NO: 22) 3′DppV/myc 5′GAA TTC TTA TCA CTC GAG GTC TTCTTC GGA AAT CAA CTT CTG TTC CGG GAC AAC GGT GTC CTC CAG GCT GAC GGC GTTGGG G 3′ (SEQ ID NO: 23) 5′Fos-1/SalI 5′GTC GAC ATG GCC CTC GAC AAG GAGC 3′ (SEQ ID NO: 24) 3′Fos-1/myc 5′CTG CAG TTA TCA CTC GAG GTC TTC TTCGGA AAT CAA CTT CTG TTC CAT CTT GGA TGA TTC CTC AAA AGC TGC CAC CAT CCGCGC TTC AGC CGC 3′ (SEQ ID NO: 25) 5′Hemo/Bam 5′GGA TTC ATG GCA TCG GTCCAA GCT TAC GCA CAG TGG 3′ (SEQ ID NO: 26) 3′ Hemo/myc 5′GAA TTC TTA TCACTC GAG GTC TTC TTC GGA AAT CAA CTT CTG TTC ACA GTT GCC AAT GGC ACC ACCATA CTT GTT CCA CGT TCC AAT TTC GAC CCA G 3′ (SEQ ID NO: 27) 5′RodA/SalI5′GTC GAC ATG AAG TTC TCT TTG AGC GCT GCT GTC CTC GC 3′ (SEQ ID NO: 28)3′RodA/myc 5′GAA TTC TTA TCA CTC GAG GTC TTC TTC GGA AAT CAA CTT CTG TTCCAG GAT AGA ACC AAG GGC AAT GCA AGG AAG ACC CAG TCC AAT GAG GG 3′ (SEQID NO: 29) PCR primers

TABLE 4 PCR primers 5′ CDA/Bam GGA TCC ATG TCT CCT ACC ATG AAG AAA CGTGCG (SEQ ID NO: 30) 3′ CDA/myc GAA TTC TTA TCA CTC GAG GTC TTC TTC GGAAAT CAA CTT CTG TTC GCA TTG CCA AGA GCT ATC CCT CTC TCC AGG GCA ATC AACCCA TTC G (SEQ ID NO: 31) 5′ CnFos/Sal GTC GAC ATG TCC CTC CCC GAT GCCTAC CCT CCG GTC ATA GCC ACC (SEQ ID NO: 32) 3′ CnFos/myc GAA TTC TTA TCACTC GAG GTC TTC TTC GGA AAT CAA CTT CTG TTC ACT TCT GTT GGC CAT TTC AGCTTC CTG TCT CGC C (SEQ ID NO: 33) 5′ Lac/Bam GGA TCC ATG GAG GAG ACT GGCAAG TCG CCA ACC GCG (SEQ ID NO: 34) 3′ Lac/myc GAA TTC TTA TCA CTC GAGGTC TTC TTC GGA AAT CAA CTT CTG TTC CGC CTC TCG AGC ACC TCT ACG TTT AACGAC CTT CCC TTT CAC TTC GCG CAG (SEQ ID NO: 35) 5′ Plb/Bam GGA TCC ATGGCT GTT CCT CCC GAG ACT CCG CGG (SEQ ID NO: 36) 3′ Plb/myc GAA TTC TTATCA CTC GAG GTC TTC TTC GGA AAT CAA CTT CTG TTC AGT GAG AGA GCG CAT ACCATT GAG CAA CAT TTC GTT GGC (SEQ ID NO: 37) 5′ TRR/Bam GGA TCC ATG CACTCC AAG GTT GTT ATC ATC GGC TCT GG (SEQ ID NO: 38) 3′ TRR/myc GAA TTCTTA TCA CTC GAG GTC TTC TTC GGA AAT CAA CTT CTG TTC CTC CTT GTC AGT GCCGAA GTA GTG CTC GGC AGG GAC ATG CAC ATC TTC GGT CTG G (SEQ ID NO: 39)5′ URE/Bam GGA TCC ATG CAT CTC CTC CCG AGA GAA ACG (SEQ ID NO: 40)3′ URE/myc GTC GAC TTA TCA CTC GAG GTC TTC TTC GGA AAT CAA CTT CTG TTCGTA AAC GAA GTA TCT CCT GGT CAA TGG GAG TTT GTC CGC GGG TGG GAC (SEQ IDNO: 41) PCR primersAll of the COMPOSITIONS and METHODS disclosed and claimed herein may bemade and executed without undue experimentation in light of the presentdisclosure. While the COMPOSITIONS and METHODS have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variation may be applied to the COMPOSITIONS and METHODSand in the steps or in the sequence of steps of the METHODS describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

1. A composition comprising: a) non-viable yeast cells; and b) anantigen from Aspergillus hemolysin; wherein the composition isformulated for administration to a subject, and wherein the compositioninduces a cell-mediated immune response in a subject.
 2. The compositionof claim 1, wherein the yeast cells were transformed to express theantigen.
 3. The composition of claim 1, wherein the yeast cells are fromSaccharomyces cerevisiae.
 4. A composition comprising: a) non-viableyeast cells; and b) an antigen from Coccidioides antigen 2 (Ag2);wherein the composition is formulated for administration to a subject,and wherein the composition induces a cell-mediated immune response in asubject.
 5. The composition of claim 4, wherein the yeast cells weretransformed to express the antigen.
 6. The composition of claim 4,wherein the yeast cells are from Saccharomyces cerevisiae.
 7. Acomposition comprising: a) non-viable yeast cells; and b) an antigenfrom Cryptococcus laccase (Lac) or chitin deacetylase (CDA); wherein thecomposition is formulated for administration to a subject, and whereinthe composition induces a cell-mediated immune response in a subject. 8.The composition of claim 7, wherein the yeast cells were transformed toexpress the antigen.
 9. The composition of claim 7, wherein the yeastcells are from Saccharomyces cerevisiae.
 10. A method to reduceAspergillus fungal burden or treat Aspergillus fungal infection in asubject, comprising administering to the subject the composition ofclaim
 1. 11. A method to reduce Coccidiodes fungal burden or treatCoccidiodes fungal infection in a subject, comprising administering tothe subject the composition of claim
 4. 12. A method to reduceCryptococcus fungal burden or treat Cryptococcus fungal infection in asubject, comprising administering to the subject the composition ofclaim 7.